Compositions, methods and uses of alpha 1-antitrypsin for early intervention in bone marrow transplantation and treatment of graft versus host disease

ABSTRACT

Embodiments of the present invention relate to compositions and methods for treatment of subjects in need of or having a bone marrow transplant. Certain embodiments describe compositions and methods for treatment of conditions associated with bone marrow transplantations in a subject, for example, Graft versus Host Disease (GvHD) or bone marrow transplantation rejection. Some embodiments concern early or immediate bone marrow transplantation rejection. Certain embodiments relate to compositions and uses of alpha1-antitrypsin (α1-antitrypsin, AAT) and carboxyterminal peptide derivatives thereof and/or compositions and uses of serine protease inhibitors, immunomodulators or anti-inflammatory agent activity similar to that of AAT.

PRIORITY

This Application is a divisional of U.S. application Ser. No. 13/209,349which is a continuation of U.S. application Ser. No. 11,916,521 filedDec. 4, 2007, which is a national stage application of PCT ApplicationNo. PCT/US2006/22436, filed Jun. 7, 2006, which claims priority to U.S.Provisional Application No. 60/687,580 filed Jun. 7, 2005. All priorapplications are incorporated herein by reference in their entirety forall purposes.

FIELD

Embodiments of the present invention relate to compositions and methodsfor treatment of subjects in need of or having a bone marrow transplant.Certain embodiments describe compositions and methods for treatment ofconditions associated with bone marrow transplantations in a subject,for example, Graft versus Host Disease (GvHD) or bone marrowtransplantation rejection. Some embodiments concern early or immediatebone marrow transplantation rejection. Certain embodiments relate tocompositions and uses of alpha1-antitrypsin (α1-antitrypsin, AAT) andcarboxyterminal peptide derivatives thereof and/or compositions and usesof serine protease inhibitors, immunomodulators or anti-inflammatoryagent activity similar to that of AAT.

BACKGROUND Serine Proteases

Serine proteases serve an important role in human physiology bymediating the activation of vital functions. In addition to their normalphysiological function, serine proteases have been implicated in anumber of pathological conditions in humans. Serine proteases arecharacterized by a catalytic triad consisting of aspartic acid,histidine and serine at the active site.

Naturally occurring serine protease inhibitors have been classified intofamilies primarily on the basis of the disulfide bonding pattern and thesequence homology of the reactive site. Serine protease inhibitors,including the group known as serpins, have been found in microbes, inthe tissues and fluids of plants, animals, insects and other organisms.At least nine separate, well-characterized proteins are now identified,which share the ability to inhibit the activity of various proteases.Several of the inhibitors have been grouped together, namelyα1-antitrypsin-proteinase inhibitor, secretory leukocyte proteaseinhibitor or SLPI, antithrombin III, antichymotrypsin, C1-inhibitor, andα2-antiplasmin, which are directed against various serine proteases,i.e., leukocyte elastase, thrombin, cathepsin G, chymotrypsin,plasminogen activators, and plasmin. These inhibitors are members of theα1-antitrypsin-proteinase inhibitor class. The protein α2-macroglobulininhibits members of all four classes of endogenous proteases: serine,cysteine, aspartic, and metalloproteases. However, other types ofprotease inhibitors are class specific. For example, theα1-antitrypsin-proteinase inhibitor (also known as (α1-antitrypsin orAAT) and inter-alpha-trypsin inhibitor inhibit only serine proteases,α1-cysteine protease inhibitor inhibits cysteine proteases, andα1-anticollagenase inhibits collagenolytic enzymes of the metalloenzymeclass.

The normal plasma concentration of ATT ranges from 1.3 to 3.5 mg/mlalthough it can behave as an acute phase reactant and increase 3-4-foldduring host response to inflammation and/or tissue injury such as withpregnancy, acute infection, and tumors. It easily diffuses into tissuespaces and forms a 1:1 complex with target proteases, principallyneutrophil elastase. Other enzymes such as trypsin, chymotrypsin,cathepsin G, plasmin, thrombin, tissue kallikrein, and factor Xa canalso serve as substrates. The enzyme/inhibitor complex is then removedfrom circulation by binding to serpin-enzyme complex (SEC) receptor andcatabolized by the liver and spleen. ATT appears to represent animportant part of the defense mechanism against activity by serineproteases.

α1-antitrypsin is one of few naturally occurring mammalian serineprotease inhibitors currently approved for the clinical therapy ofprotease imbalance. Therapeutic α1-antitrypsin has been commerciallyavailable since the mid 1980's and is prepared by various purificationmethods (see for example Bollen et al., U.S. Pat. No. 4,629,567;Thompson et al., U.S. Pat. Nos. 4,760,130; 5,616,693; WO 98/56821).Prolastin is a trademark for a purified variant of α1-antitrypsin and iscurrently sold by Talectris/Grifols Company (U.S. Pat. No. 5,610,285Lebing et al., Mar. 11, 1997). Recombinant unmodified and mutantvariants of α1-antitrypsin produced by genetic engineering methods arealso known (U.S. Pat. No. 4,711,848); methods of use are also known,e.g., (α1-antitrypsin gene therapy/delivery (U.S. Pat. No. 5,399,346).

Graft Rejection

There are many diseases that culminate in organ dysfunction or failure.Representative non-limiting examples include renal failure due todiabetes melitus, hypertension, urinary output obstruction, drug-inducedtoxicity, or hypoperfusion, as well as cardiac dysfunction due toischemic coronary artery disease, cardiomyopathy/infection, orvalvulopathy. Pulmonary diseases include substantial damage due tochronic obstructive pulmonary disease (COPD, including chronicbronchitis and emphysema), AAT deficiency, cystic fibrosis, andinterstitial fibrosis. Under certain conditions, the only therapeuticoption for treatment of a subject may be organ transplantation.Pancreatic-islet transplantation provides diabetic patients with theonly option for a tightly-controlled blood glucose level, as proven tobe essential for prevention of diabetic complications. In the case ofislets, post-transplant inflammation, which precedes immune rejection,is a critical determinant of graft survival. This early inflammation ismediated by cells other than the impending allospecific immune cells.

One challenge to therapeutic transplantation is the damaging effects ofthe host immune system on the transplant. MHC molecules exist on thesurfaces of cells and the particular structures of MHC molecules aretypically unique for each individual (with the exception of identicaltwins, where the MHC molecule complements are identical). The immunesystem is programmed to attack foreign or “non-self” MHC-bearingtissues. For these reasons, when an organ or tissue is transplanted intoa recipient, an effort is made to optimize the degree of tissue matchingbetween donor and recipient. MHC antigens are characterized for therecipient and donors. Matching a donor to an allograft recipient by MHCstructure reduces the magnitude of the rejection response. An archetypalexample is blood group matching. Most transplants are allografts thatoccur between non-identical members of the same species. Since thesematches are imperfect, there is an expected graft rejection immuneresponse associated with allografts. Current methods used, in order toenhance graft survival, include medications to suppress the immuneresponse which can result in graft rejection. These medications arereferred to immunosuppressant or antirejection drugs, such asprednisone, cyclosporine A, and cyclophosphamide, to name a few. Asmentioned above, local inflammation is experienced immediately aftergrafting, and cells that are particularly sensitive to non-specificinflammation, such as islets, can endure graft dysfunction more severelythan other types.

Despite advances in the field of antirejection therapy, graftmaintenance remains a challenge since the available antirejectiontherapies are imperfect. For example, immunosuppression enhances therisk for opportunistic infection or neoplasia. Toxicities abound andinclude, but are not limited to, diabetes, organ dysfunction, renalfailure, hepatic dysfunction, hematological defects, neuromuscular andpsychiatric side effects, and many others. Therefore, there is a needfor a more effective anti-rejection medical treatment that prolong graftsurvival and improve the quality of life.

Bone marrow transplantation is a unique kind of transplant where immunecells from a donor are transferred into a recipient, thereby conferringthe donor immune system into the recipient. Here, the graft is capableof generating an immune response against the host, and this is termed“graft versus host” disease (GvHD). Immunosuppressive and antimicrobialtreatment is required to block adverse consequences of GvHD, and a needexists for safer and more effective inhibitors of the adverse effects bythe graft. In certain embodiments, a subject undergoing a bone marrowtransplantation can be administered a therapeutically effective amountof AAT or carboxyterminal derivative thereof, to treat a subject earlyor immediately after bone marrow transplantation. In some embodiments, atherapeutically effective amount of AAT or carboxyterminal derivativethereof, can be used to treat a subject having GvHD or suspected ofdeveloping GvHD wherein the treatment reduces the incidence of orprevents mortality of a subject.

Because of some of the difficulties and inadequacies of conventionaltherapy for treating transplantation complications and associatedside-effects, new therapeutic modalities are needed.

SUMMARY

Embodiments of the present invention provide for methods for treating asubject having or in need of a transplant. In accordance with theseembodiments, a subject may be treated with a composition for reducingthe risk of a transplant rejection or a side-effect of a transplantrejection in a subject. In accordance with this method the subject canbe administered a composition including a compound that is capable ofsignificantly reducing serine protease activity. The composition may beadministered before transplantation, during transplantation, aftertransplantation or combination thereof. In addition, the composition mayfurther include one or more anti-transplant rejection agent,anti-inflammatory agent, immunosuppressive agent, immunomodulatoryagent, anti-microbial agent, or a combination thereof.

In certain embodiments of the invention, a composition capable ofsignificantly reducing serine protease activity can includealpha-1-antitrypsin, an analog thereof or a combination thereof. Atransplant of the present invention may include an organ transplantand/or a non-organ transplant. For example lung, kidney, heart, liver,cornea, skin, stem cells, soft tissue (e.g. facial componenttransplant), intestinal transplants, bone marrow, pancreatic islet,pancreas transplant or combination thereof are contemplated.

Embodiments of the present invention provide for methods forameliorating symptoms or signs experienced by a subject having or inneed of a transplant. In accordance with these embodiments, symptoms orsigns may include conditions associated with graft versus host disease(GVHD), or graft rejection. In one example, methods disclosed herein maybe used to treat a subject undergoing bone marrow transplantation. Inanother embodiment, symptoms or signs may include but is not limited toone or more of the following, kidney failure, lung failure, heartfailure, malaise, fever, dry cough, anorexia, weight loss, myalgias, andchest pains, ventilatory compromise, sweating, nausea, vomiting, fever,abdominal pain, bloody diarrhea, mucosal ulcerations, reduced renalfunction (increased creatinine, decreased urine output), reducedpulmonary function (increased shortness of breadth, fever, cough,sputum, hypoxemia), reduced cardiac function (shortness of breach, chestpain, fatigue, pulmonary or peripheral edema, valvulopathy), reducedislet function (increased glucose, diabetes melitus), graft versus hostdisease (gastrointestinal (GI) ulceration, pulmonary failure, skinulceration, coagulopothy, CNS dysfunction (mental status changes, coma)CMV (cytomeglovirus infection, viral, fungal parasitic infection)).

In some embodiments, a subject in need of a bone marrow transplant canbe administered AAT or a carboxyterminal derivative of AAT before,during, or after bone marrow transplantation. In certain embodiments,bone marrow cells can be pretreated with a composition of AAT or acarboxyterminal derivative of AAT prior to introduction to a subject. Inother embodiments, any cellular transplant matter can be pre-treatedwith a composition of AAT or a carboxyterminal derivative of AAT priorto introduction to a subject in need of a cellular transplant. Certainembodiments concern treating a subject with compositions disclosedherein in order to prevent a subject from developing GvHD. In accordancewith these embodiments, a subject can be treated early or immediatelyafter bone marrow transplantation. In other embodiments, a subjecthaving had bone marrow transplantation demonstrating symptoms or signsof GvHD as recognized in the art can be given a composition of AATand/or a carboxyterminal derivative of AAT in order to reduce or preventmortality of the subject.

In certain embodiments, administration of a composition comprising AATand/or carboxyterminal derived peptides of AAT reduced serum levels ofproinflammatory cytokines in allogeneic recipients compared to a controlnot receiving the compositions . It was demonstrated that AAT treatmentreduced the expansion of alloreactive T effector cells but enhanced therecovery of regulatory T cells (Tregs). In certain embodiments, AATcompositions were capable of altering the ratio of donor T effector to Tregulatory cells in favor of reducing the pathological process. However,despite altering the ratio in vivo, AAT had no direct effects on eitherthe donor T effector cells or Tregs in vitro. In contrast, AATsuppressed LPS-induced in vitro secretion of pro-inflammatory cytokinessuch as TNFα and IL-1β, enhanced the production of the anti-inflammatorycytokine IL-10 and impaired NFκB translocation in the host dendriticcells. In light of its long history of safety in humans, these findingssuggest that administration of AAT represents a novel and viablestrategy to mitigate clinical GvHD.

In other embodiments, it is contemplated that specific interleukins suchas IL-32 can be inhibited in a subject in need of such a therapy.Compositions disclosed herein can be used to inhibit IL-32 in a subjecthaving increased activity of this and other cytokines.

It is contemplated herein that AAT can include naturally occurring AATharvested from human or other mammalian plasma and/or commerciallyavailable formulations such as Aralast™, Zemaira™, Kamada's agents andProlastin™ and ProlastinC.™ Therapeutically effective doses of AAT caninclude those doses administered to AAT deficient patients or in a rangeof about 1 to about 100 mg/kg in a single or multiple dose regimen. Itis contemplated herein that AAT or derivative thereof can beadministered to a subject in need thereof and blood can be drawn fromthe subject in order to assess the level of AAT in the subject. Inaddition, once the level of AAT is determined, a health professional mayadminister more or less AAT to the subject depending on need.

Embodiments of the present invention provide methods for promotingprolonged graft survival and function in a subject includingadministering to a subject in need thereof a therapeutically effectiveamount of a composition including a substance exhibiting α1-antitrypsinor α1-antitrypsin analog or inhibitor of serine protease activity or afunctional derivative thereof.

Embodiments of the present invention provide for methods for treating asubject in need of an immunotolerance therapy. In accordance with theseembodiments, a subject may be treated with a composition for reducingthe risk of a dysfunctional immune responses or a side-effect of adysfunctional immune response in a subject. In another embodiment,methods herein provide for inducing immune tolerance specific for agraft and/or reduce the need for immunosuppressive therapy. Inaccordance with this embodiment, the immune system of the transplantrecipient may have reduced or lost the specific ability to attack thegraft while maintaining its ability to mount any other type of immuneattack. In accordance with this method the subject can be administered acomposition including a compound that is capable of significantlyreducing serine protease activity or other activity associated withα1-antitrypsin or α1-antitrypsin analog. In certain embodiments, acomposition capable of significantly reducing serine protease activitycan include alpha-1-antitrypsin, an analog thereof or a combinationthereof. In accordance with these embodiments, one example forimmunotolerance therapy can include inhibiting cytokine production.

Embodiments of the present invention provide for methods for reducingTNFα (tumor necrosis factor alpha) levels in a subject includingadministering a composition including alpha-1-antitrypsin, an analogthereof or a combination thereof to a subject in need of such atreatment.

Embodiments of the present invention provide for methods for treating asubject in need of an immunotolerance therapy. In accordance with theseembodiments methods are provided for reducing NO production and/orreducing apoptosis and/or inhibiting cytomegleovirus (infection andreactivation) including administering a composition including a compoundthat is capable of significantly reducing serine protease activityand/or other alpha-1-antitrypsin activity. In certain embodiments of theinvention, a composition capable of significantly reducing serineprotease activity and/or mimicking other alpha-1-antitrypsin activitycan include alpha-1-antitrypsin, an analog thereof, or a combinationthereof.

In certain embodiments of the present invention, the anti-inflammatorycompound or immunomodulatory drug can include but is not limited to oneor more of interferon, interferon derivatives including betaseron,beta-interferon, prostane derivatives including iloprost, cicaprost;glucocorticoids including cortisol, prednisolone, methyl-prednisolone,dexamethasone; immunsuppressives including cyclosporine A, FK-506,methoxsalene, thalidomide, sulfasalazine, azathioprine, methotrexate;lipoxygenase inhibitors comprising zileutone, MK-886, WY-50295,SC-45662, SC-41661A, BI-L-357; leukotriene antagonists; peptidederivatives including ACTH and analogs thereof; soluble TNF-receptors;TNF-antibodies; soluble receptors of interleukins, other cytokines,T-cell-proteins; antibodies against receptors of interleukins, othercytokines, T-cell-proteins; and calcipotriols; Celcept®, mycophenolatemofetil, and analogues thereof taken either alone or in combination.

Embodiments of the present invention provide for methods for reducinggraft rejection in a subject. In accordance with these embodiments, asubject may be treated with a composition for reducing the risk of graftrejection responses or a side-effect of a graft rejection response in asubject. In accordance with this method, the subject can be administereda composition including a compound that is capable of significantlyreducing serine protease activity. In certain embodiments, a compositioncapable of significantly reducing serine protease activity can includeα1-antitrypsin, an analog thereof or a combination thereof. In oneexample, reducing graft rejection may include reducing the symptomsassociated with graft rejection in a subject having an organ transplant,such as a kidney transplant or a bowel transplant or a non-organtransplant, such as a bone marrow transplant soft tissue transplant.

In yet another embodiment, the present invention may include combinationtherapies including compositions exhibiting α1-antitrypsin, an analogthereof, or substance with serine protease inhibitor activity. Forexample, a composition may include α1-antitrypsin and another serineprotease inhibitor administered simultaneously or in separatecompositions.

In accordance with embodiments disclosed herein, any of the disclosedcompositions may be used to ameliorate symptoms associated with atransplant. These symptoms may include but are not limited to,infiltration of graft with cells and/or serum factors (for example,complement, anti-graft antibodies), increased cytokine and/or chemokineproduction, increased nitric oxide production, increased apoptosis andcell death, and increased immune response against the transplant tissueand/or cells.

In another aspect, the present invention provides for a method ofameliorating a symptom or sign associated with transplantation in asubject in need of said amelioration. In accordance with thisembodiment, a composition may be administered to a subject such as apharmaceutically effective amount of a substance of α1-antitrypsin, ananalog thereof or serine protease inhibitor activity, wherein thecomposition is capable of reducing, preventing or inhibiting serineprotease or protease activity and/or binds to the sec receptor or otheractivity.

In certain embodiments, synthetic and/or naturally occurring peptidesmay be used in compositions and methods of the present invention forexample, providing serine protease inhibitor activity. Homologues,natural peptides, with sequence homologies to AAT including peptidesdirectly derived from cleavage of AAT may be used or other peptides suchas, peptides that inhibit serine proteases or have AAT-like activity.Other peptidyl derivatives, e.g., aldehyde or ketone derivatives of suchpeptides are also contemplated herein. Without limiting to AAT andpeptide derivatives of AAT, compounds like oxadiazole, thiadiazole andtriazole peptoids and substances comprising certain phenylenedialkanoateesters, CE-2072, UT-77, and triazole peptoids may be used. Examples ofanalogues are TLCK (tosyl-L-lysine chloromethyl ketone) or TPCK(tosyl-L-phenylalanine chloromethyl ketone).

In other embodiments, an agent that reduces the occurrence of graftrejection, promotes prolonged graft function or promotes prolongedallograft survival can also be an inhibitor of serine protease activity,an inhibitor of elastase, or an inhibitor of proteinase-3. An inhibitorof serine protease activity can include, but is not limited to, smallorganic molecules including naturally-occurring, synthetic, andbiosynthetic molecules, small inorganic molecules includingnaturally-occurring and synthetic molecules, natural products includingthose produced by plants and fungi, peptides, variants ofα1-antitrypsin, chemically modified peptides, and proteins.

In some embodiments, AAT peptides contemplated for use in thecompositions and methods of the present invention are also intended toinclude any and all of those specific AAT peptides other than the 10amino acid AAT peptides of SEQ ID NO. 1 depicted supra. Any combinationof consecutive amino acids depicting a portion of AAT or AAT-likeactivity may be used, such as amino acids 2-12, amino acids 3-13, 4-14,etc. of SEQ ID NO. 1, as well as any and all AAT peptide fragmentscorresponding to select amino acids of SEQ ID NO. 1. Applicants areherein entitled to compositions based upon any and all AAT peptidevariants based upon the amino acid sequence depicted in SEQ ID NO. 1.

In one aspect of the invention, the pharmaceutical compositions of thepresent invention are administered orally, systemically, via an implant,intravenously, topically, intrathecally, intratracheally,intracranially, subcutaneously, intravaginally, intraventricularly,intranasally such as inhalation, mixed with grafts by flushing of organor suspension of cells, or any combination thereof.

As such, those skilled in the art will appreciate that the conception,upon which this disclosure is based, can readily be used as a basis fordesigning other methods for carrying out the several features andadvantages of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain embodiments of the presentinvention. The embodiments may be better understood by reference to oneor more of these drawings in combination with the detailed descriptionof specific embodiments presented herein.

FIGS. 1A-1D illustrates an exemplary method of treating islet allograftswith AAT. Islets from DBA/2 mice (H-2d) were transplanted under therenal capsule of streptozotocin-induced hyperglycemic C57BL/6 mice(H-2b). (A) Glucose levels from days 6-18. (B) Treatment protocols.Control and full AAT treatment are described in panel A. Early AATtreatment consists of treatment on days −1, 1 and 3 (2 mg, n=3). LateAAT treatment consists of treatment from day 2 and on every 2 days (2mg, n=3). (C) Effect of mouse anti-human-AAT antibodies. Dashed lineindicates post transplantation glucose levels of a mouse under full AATtreatment protocol (see A, B) that was immunized by multipleadministrations of human AAT prior to transplantation (1 representative,n=3). Solid line indicates glucose levels of a non-immunized mousetreated under full AAT treatment protocol (1 representative, n=10).Arrow indicates detection of treatment-induced, anti-human-AATantibodies in the non-immunized representative mouse. (D) Comparison ofday 15 post-transplantation glucose levels in mice that were under fulltreatment protocol with ALB (n=3) or AAT (non-immunized n=10, immunizedn=3). Of the AAT-treated group, antibodies were detected on day 15 in3/3 immunized mice and in 6/10 non-immunized mice.

FIGS. 2A-2D illustrates an exemplary method of the effect of AAT onthioglycolate-elicited peritoneal cellular infiltrates. (A) Total cellpopulation of lavaged cells of (o) saline or (Δ) AAT-treated (5 mg)thioglycolate-injected mice. (B) Percent cell population fromsaline-treated mice at 48 hours. (C) Oxidation of AAT. (D)Identification of elicited macrophages and neutrophils.

FIGS. 3A-3C illustrates an exemplary method of the effect of AAT onMHC-incompatible, NIH-3T3-fibroblast-elicited peritoneal cellularinfiltrates. (A) Cell numbers. The number of cells in each subpopulationwas calculated from the percentages obtained by FACS analysis, and totalnumber of cells in the infiltrate. (B) Representative FACS analysis. (C)Effect of AAT on intensity and function of infiltrate elicited by isletallograft. Left, Hematoxilyn and Eosin (H&E) staining of day 7 isletallografts. Right, Immunohistochemistry (IHC) with anti-insulinantibodies of day 15 islet grafts. R, renal parenchyma, G, graft, C,renal capsule.

FIGS. 4A-4H illustrates an exemplary method of the effect of AAT onislet responses. (A-D) Mean±SEM of A. nitric levels, B. Cell viabilityand C. MIP-1α levels. Dashed line represents islets incubated atone-30th the concentration of IFNγ/IL-1β. TNFα levels. (E) Insulininduction assay. (F) Streptozotocin toxicity. Each image depicts arepresentative islet from one pancreas. (G) Cellular content of islets.(H) MHC class II expression.

FIGS. 5A-5D illustrates the effect of AAT on TNFα. (A) Islets fromC57BL/6 mice were cultured (100 islets/well in triplicate) in thepresence of AAT (0.5 mg/ml) or TACE inhibitor (10 mM) 1 hour beforestimulation by IFNγ (5 ng/ml) plus IL-1β (10 ng/ml). Left, mean±SEMchange in TNFα in supernatants after 72 hours of incubation. Right,mean±SEM fold change in membrane TNFα on islet cells after 5 hours ofincubation, according to FACS analysis. (B) Representative FACS analysisof membrane TNFα on stimulated islet cells in the absence (open area) orpresence (shaded area) of AAT. (C) Streptozotocin-induced hyperglycemia.

FIGS. 6A-6D illustrates the effect of AAT on Islet allografttransplantation. 6A illustrates the time course study aftertransplantation. 6B illustrates an immune infiltrate found outside thegraft area. 6C illustrates an increase in the presence of CD4+ and acomparative decrease in monocytes and neutrophils. 6D illustrates levelsof glucose reflecting a level of tolerance with respect to daysfollowing allografting of the same donor (left) and a 3^(rd) donorre-graft (right), indicating induction of specific immune tolerance.

FIGS. 7A-7E illustrates the production of AAT by islet cell andreflection of islet graft survival. 7A illustrates a time courseexpression of mouse AAT mRNA after cytokine production (IL-1β and IFNγ)(left) and at 8 hours (right). 7B illustrates an example of islet injuryduring pancreatitis; the histology of normal islets (top left), thehistology of islets of an inflamed pancreas (top right) and expressionof mouse AAT in islets obtained from the pancreata in an acutepancreatitis model (bottom). 7C illustrates an example of samples ofislet allografts taken post grafting and the percent change in AAT mRNAlevels were assessed. 7D illustrates an example of islet protection fromcytokine injury with endogenous AAT by introducing oncostatin M (aninterleukin 6 (IL-6) family member) that induces AAT expression inislets, oncostatin M and AAT levels (top left); nitric oxide andviability levels assessed (top right) and nitric oxide productionrepresenting islet viability after 4 day exposure to oncostatin M andAAT production decreasing cytokine effects on the islets (bottom).

FIGS. 8A-8D illustrates the effect of AAT on human islets and theproduction of nitric oxide (8A), TNF-α production (8B) IL-6 (8C) andIL-8 (8D).

FIGS. 9A-9C represents affects of AAT to reduce mortality in bone marrowtransplantations in a mouse model under various conditions. A)represents percent survival of mice after bone marrow transplantation(BMT). B) represents BMT transplanted mice under various conditionsrelated to T cell depletion. C) represents BMT mouse model having T-celldepleted bone marrow and followed under various conditions .

FIGS. 10A-10D represent affects of human AAT (hAAT) and a control on theratio of donor Teff:Treg cells in a subject Each point represents oneindividual mouse (n=2−5/group). (A) CD4⁺: Treg ratio on day +21, vehiclevs. hAAT, P=0.037 (B) CD8⁺:Treg ratio on day+21, vehicle vs. hAAT,P=0.022. (C) CD4⁺:Treg ratio on day+28, vehicle vs. hAAT, P=0.0223 and(D) CD8⁺:Treg ratio on day+28, vehicle vs. hAAT, P=0.0127.

FIG. 11 represents affects of AAT on proinflammatory cytokines producedafter BMT.

FIGS. 12A and 12B represent a histogram plot (A) of some affects ofinterleukin-32 (IL-32) and a control on cytokine expression and aWestern blot (B) representative of affects of various concentrations ofAAT on IL-32 (β and γ forms)

FIGS. 13A-13D represent a Western Blot (A) and histogram plots (B-D) ofAAT affects on proliferation and TNF secretion of mixed lymphocytecultures (MLCs)

FIGS. 14A-14E represent effects of AAT on GvHD severity and mortalitywhere (A) represents the aAT treatment regimen, (B) represents survival,(C) represents severity of GvHD, (D) represents change in body weightover time and (E) represents donor chimerism.

FIGS. 15A-15D represent effect of AAT on cytokine RNA and proteinexpression after BMT. A) represents various cytokine plots and B) C) andD) represent cytokine plasma levels at days 3, 7 and 10 after BMT.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS Definitions

Terms that are not otherwise defined herein are used in accordance withtheir plain and ordinary meaning.

As used herein, “a” or “an” may mean one or more than one of an item.

As used herein “analog of alpha-1-antitrypsin” may mean a compoundhaving alpha-1-antitrypsin-ike activity. In one embodiment, an analog ofalpha-1-antitrypsin is a functional derivative of alpha-1-antitrypsin.In a particular embodiment, an analog of alpha-1-antitrypsin is acompound capable of significantly reducing serine protease activity. Forexample, an inhibitor of serine protease activity has the capability ofinhibiting the proteolytic activity of trypsin, elastase, kallikrein,thrombin, cathepsin G, chymotrypsin, plasminogen activators, plasminand/or other serine proteases.

As used herein “immunomodulatory drugs or agents”, it is meant, e.g.,agents which act on the immune system, directly or indirectly, e.g., bystimulating or suppressing a cellular activity of a cell in the immunesystem, e.g., T-cells, B-cells, macrophages, or antigen presenting cells(APC, dendritic cells), or by acting upon components outside the immunesystem which, in turn, stimulate, suppress, or modulate the immunesystem, e.g. cytokines, e.g., hormones, receptor agonists orantagonists, and neurotransmitters; immunomodulators can be, e.g.,immunosuppressants or immunostimulants.

It is to be understood that the terminology and phraseology employedherein are for the purpose of description and should not be regarded aslimiting

DETAILED DESCRIPTION OF THE INVENTION

In the following sections, various exemplary compositions and methodsare described in order to detail various embodiments of the invention.It will be obvious to one skilled in the art that practicing the variousembodiments does not require the employment of all or even some of thespecific details outlined herein, but rather that concentrations, timesand other specific details may be modified through routineexperimentation. In some cases, well known methods or components havenot been included in the description.

Embodiments of the present invention provide for methods for treating asubject having or in need of a transplant. In accordance with theseembodiments, a subject may be treated with a composition capable ofsignificantly reducing serine protease activity. In addition, oneembodiment of the present invention provides for methods includingtreating a subject with a composition comprising a compound havingα-1-antitrypsin activity. In one embodiment, the composition can includeα-1-antitrypsin, analog thereof or a serine protease inhibitor to forexample, promote transplant survival or reduce a side effect of thetransplant. Further, the administration of the composition can be beforetransplantation, during transplantation, after transplantation orcombination thereof. In addition, the composition may further includeone or more additional therapies such as immunosuppressive therapies. Atransplant of the present invention may include transplantation of anorgan such as lung, kidney, heart, liver, skin, pancreas, or bowel organor non-organ such bone marrow, pancreatic islet, cornea, and/or softtissue.

Serine protease inhibitors, have been found in a variety of organisms.At least nine separate, well-characterized proteins are now identified,which share the ability to inhibit the activity of various proteases.Several of the inhibitors have been grouped together, such as theα₁-antitrypsin-proteinase inhibitor. Serine proteases include but arenot limited to leukocyte elastase, thrombin, cathepsin G, chymotrypsin,plasminogen activators, and plasmin.

Embodiments of the present invention provide for methods for promotingtransplantation, graft survival, reducing graft rejection and/orreducing or preventing side-effects associated with graft rejection. Inaccordance with these embodiments, side-effects may include conditionsassociated with graft versus host disease (GVHD), or graft rejection. Inone example, methods disclosed herein may be used to treat a subjectundergoing bone marrow transplantation. In another embodiment, symptomsor signs may include but is not limited to one or more of the following,malaise, fever, dry cough, myalgias, and chest pains, ventilatorycompromise, sweating, nausea, vomiting, fever, abdominal pain, bloodydiarrhea, mucosal ulcerations, reduced renal function (increasedcreatinine, decreased urine output), reduced pulmonary function(increased shortness of breadth, fever, cough, sputum, hypoxemia),reduced cardiac function (shortness of breach, chest pain, fatigue,pulmonary or peripheral edema, valvulopathy), reduced islet function(increased glucose, diabetes mellitus), graft versus host disease(gastrointestinal (GI) ulceration, pulmonary failure, skin ulceration).

Embodiments of the present invention provide for methods for treating asubject in need of an immunotolerance therapy. In accordance with theseembodiments, a subject may be treated with a composition for inducingimmune tolerance. This achieved while reducing the risk of adysfunctional immune responses or a side-effect of a dysfunctionalimmune response in a subject as typically encountered during standardimmune suppression. For example, a dysfunctional immune response may bean effect of graft rejection, pneumonia, sepsis, fungal infection,cancer. In accordance with this method the subject can be administered acomposition including a compound that is capable of significantlyreducing serine protease activity or other activity associated withα1-antitrypsin or α1-antitrypsin analog. In certain embodiments, acomposition capable of significantly reducing serine protease activitycan include α-1-antitrypsin, an analog thereof or a combination thereof.In accordance with these embodiments, one example for immunotolerancetherapy can include inhibiting cytokine production.

Any of the embodiments detailed herein may further include one or more atherapeutically effective amount of anti-microbial drugsanti-inflammatory agent, immunomodulatory agent, or immunosuppressiveagent or combination thereof

Non-limiting examples of anti-rejection agents/drugs may include forexample cyclosporine, azathioprine, corticosteroids, FK506 (tacrolimus),RS61443, mycophenolate mofetil, rapamycin (sirolimus), mizoribine,15-deoxyspergualin, and/or leflunomide or any combination thereof.

In addition, other combination compositions of methods disclosed in thepresent invention include certain antibody-based therapies. Non-limitingexamples include, polyclonal anti-lymphocyte antibodies, monoclonalantibodies directed at the T-cell antigen receptor complex (OKT3,TIOB9), monoclonal antibodies directed at additional cell surfaceantigens, including interleukin-2 receptor alpha. Antibody-basedtherapies may be used as induction therapy and/or anti-rejection drugsin combination with the compositions and methods of the presentinvention.

Embodiments of the present invention provide for methods treating asubject in need of an immunotolerance therapy. In accordance with theseembodiments, a subject may be treated with a composition capable ofsignificantly reducing serine protease activity. In one embodiment, thecomposition can include α-1-antitrypsin, analog thereof or a serineprotease inhibitor to for example, to reduce or inhibit the productionof cytokines. In accordance with these embodiments, combinationtherapies are contemplated, such as combining α-1-antitrypsincomposition with an anti-inflammatory agent.

In one particular embodiment, the present inventions provide for methodsfor reducing levels and activities of cytokines such as TNFα (tumornecrosis factor alpha). For example, the composition can includealpha-1-antitrypsin or analog thereof or combination thereof alone or incombination with other therapies. GvHD

In some embodiments, a subject in need of a bone marrow transplant canbe administered AAT or a carboxyterminal derivative of AAT before,during, or after bone marrow transplantation. In certain embodiments,bone marrow cells can be pretreated with a composition of AAT or acarboxyterminal derivative of AAT prior to introduction to a subject. Inother embodiments, any cellular transplant matter can be pre-treatedwith a composition of AAT or a carboxyterminal derivative of AAT priorto introduction to a subject in need of a cellular transplant. Certainembodiments concern treating a subject with compositions disclosedherein in order to prevent a subject from developing GvHD. In accordancewith these embodiments, a subject can be treated early or immediatelyafter bone marrow transplantation. In other embodiments, a subjecthaving had bone marrow transplantation demonstrating symptoms or signsof GvHD as recognized in the art can be given a composition of AATand/or a carboxyterminal derivative of AAT in order to reduce or preventmortality of the subject.

Acute graft-versus-host disease (GvHD) is a major complication thatprevents successful outcomes after allogeneic bone marrowtransplantation (BMT), an effective therapy for hematologicalmalignancies and other non-malignant conditions such as leukemia, severeaplastic anemia, lymphoma ,multiple myeloma, immune deficiency disorder,solid-tumor cancer, breast, cancer, ovarian cancer among others .Previous studies demonstrated that donor T cells and host antigenpresenting cells along with several proinflammatory cytokines induceGvHD and contribute to its severity. Evidence previously presenteddemonstrates that alpha-1-anti-trypsin (AAT) can reduce production ofpro-inflammatory cytokines, induce anti-inflammatory cytokines andinterfere with maturation of dendritic cells. Using well-characterizedmouse models of BMT, effects of AAT on GvHD severity have been studiedherein. Some embodiments of the present invention concern administrationof AAT early or immediately after BMT (early intervention) decreasedseverity of the disease. In other embodiments, administration of acomposition of AAT early or immediately after BMT reduces mortality in asubject having or at risk of developing GvHD. Other embodimentsdisclosed herein demonstrated that administration of AAT and/orcarboxyterminal derived peptides of AAT reduced bone marrow cellrejections if introduced soon after transplantaion.

The pathophysiology of GvHD involves donor T cell interactions with hostantigen presenting cells and the subsequent production ofproinflammatory cytokines (cytokine storm), alongside activation ofalloreactive T effector cells (T effectors) that cause target organdamage. By contrast, donor derived mature foxp3+ T regulatory cells(Tregs) can downregulate alloreactivity. Thus the ratio between donor Teffectors and donor Tregs plays a key role in the severity of GvHD.Attempts to reduce GvHD by T cell depletion have led to significantrelapse of malignancy due to the loss of graft versus leukemia (GVL),failure of engraftment, and an increase in the rate of opportunisticinfections.

In certain embodiments, administration of a composition comprising AATand/or carboxyterminal derived peptides of AAT reduced serum levels ofproinflammatory cytokines in allogeneic recipients compared to a controlnot receiving the compositions. It was demonstrated that AAT treatmentreduced the expansion of alloreactive T effector cells but enhanced therecovery of regulatory T cells (Tregs). In certain embodiments, AATcompositions were capable of altering the ratio of donor T effector to Tregulatory cells in favor of reducing the pathological process. However,despite altering the ratio in vivo, AAT had no direct effects on eitherthe donor T effector cells or Tregs in vitro. In contrast, AATsuppressed LPS-induced in vitro secretion of pro-inflammatory cytokinessuch as TNFα and IL-1β, enhanced the production of the anti-inflammatorycytokine IL-10 and impaired NFκB translocation in the host dendriticcells. In light of its long history of safety in humans, these findingssuggest that administration of AAT represents a novel and viablestrategy to mitigate clinical GvHD.

IL-32

Interleukin-32 (IL-32) was originally identified in NK cells andIL-2-activated human T-lymphocytes. As T-cells are activated inallogeneic transplantation, the role of IL-32 in human mixed lymphocytecultures (MLC) and graft-versus-host-disease (GVHD) was determined. Inallogeneic MLC, IL-32 increased 2-fold in responding T-cells,accompanied by 5-fold increases of TNFα, IL-6 and IL-8. After allogeneichematopoietic cell transplantation, IL-32 mRNA levels in bloodleukocytes were statistically significantly higher in patients withacute GVHD (n=10) than in serial samples from patients who did notdevelop acute GVHD (n=5; p=0.02). No significant changes in IL-32 levelswere present in patients with treated (n=14) or untreated (n=8) chronicGVHD, compared to healthy controls (n=8) (p=0.5 and p=0.74,respectively). As IL-32 is activated by proteinase-3 (PR3), the effectof α-1 antitrypsin (AAT) on IL-32 levels and showed suppression of IL-32and T-lymphocyte proliferation in MLC was examined. In an MHC-minorantigen disparate murine transplant model, pre- and post-conditioningtreatment with AAT resulted in attenuation or prevention of GVHD andsuperior survival compared to albumin-treated controls (e.g. 80% versus44%; p=0.04). These findings suggest that AAT modulates immune andinflammatory functions and may represent a novel approach to prevent ortreat GVHD.

In certain embodiments, it is contemplated that AAT or AAT-relatedcompositions (e.g. mutants and peptide derivatives) can prevent IL-32activation, thereby interfering with alloactivation. Although thisactivity is not restricted to IL-32, and, hence AAT will affectadditional targets, an inhibitory effect of ATT on alloactivation mightprove beneficial in the prevention or therapy of GVHD.

It is contemplated herein that AAT can include naturally occurring AATharvested from human or other mammalian plasma and/or commerciallyavailable formulations such as Aralast™, Zemaira™, Aralast™, Kamada'sagents and Prolastin™ and ProlastinC.™ Therapeutically effective dosesof AAT can include those doses administered to AAT deficient patients orin a range of about 1 to about 100 mg/kg in a single or multiple doseregimen. It is contemplated herein that AAT or derivative thereof can beadministered to a subject in need thereof and blood can be drawn fromthe subject in order to assess the level of AAT in the subject. Inaddition, once the level of AAT is determined, a health professional mayadminister more or less AAT to the subject depending on need andcircumstances of the subject.

In one embodiment, the reduction, prevention or inhibition of rejectionof transplantation or side effects thereof associated with one or moreof each of the above-recited conditions may be about 10-20%, 30-40%,50-60%, or more reduction or inhibition due to administration of thedisclosed compositions.

In one embodiment of the present invention a composition may includecompounds that engage molecules for the SEC receptor to treat a subjectundergoing a transplantation and/or in need of immunotolerance therapy.In each of the recited methods, an α1-antitrypsin (e.g. mammalianderived) or inhibitor of serine protease activity substance contemplatedfor use within the methods of the present invention can include a seriesof peptides including carboxyterminal amino acid peptides correspondingto AAT. These pentapeptides can be represented by a general formula (I):I-A-B-C-D-E-F-G-H-II (note: in the Sequence Listing F=X), wherein I isCys or absent; A is Ala, Gly; Val or absent; B is Ala, Gly, Val, Ser orabsent; C is Ser, Thr or absent; D is Ser, Thr, Ans, Glu, Arg, Ile, Leuor absent; E is Ser, Thr, Asp or absent; F is Thr, Ser, Asn, Gln, Lys,Trp or absent; G is Tyr or absent; H is Thr, Gly, Met, Met(O), Cys, Thror Gly; and II is Cys, an amide group, substituted amide group, an estergroup or absent, wherein the peptides includes 4 or more consecutiveamino acids and physiologically acceptable salts thereof. Among thisseries of peptides, several are equally acceptable including FVFLM (SEQID NO. 1), FVFAM (SEQ. ID NO. 2), FVALM (SEQ. ID NO. 3), FVFLA (SEQ. IDNO. 4), FLVFI (SEQ. ID NO. 5), FLMII (SEQ. ID NO. 6), FLFVL (SEQ. ID NO.7), FLFVV (SEQ. ID NO. 8), FLFLI (SEQ. ID NO. 9), FLFFI (SEQ. ID NO.10), FLMFI (SEQ. ID NO. 11), FMLLI (SEQ. ID NO. 12), FIIMI (SEQ. ID NO.13), FLFCI (SEQ. ID NO. 14), FLFAV (SEQ. ID) NO. 15), FVYLI (SEQ. ID NO.16), FAFLM (SEQ. ID NO. 17), AVFLM (SEQ. ID NO. 18), and any combinationthereof.

In several embodiments herein, AAT peptides contemplated for use in thecompositions and methods of the present invention are also intended toinclude any and all of those specific AAT peptides of SEQ ID NO. 1depicted supra. Any combination of consecutive amino acids simulatingAAT or AAT-like activity may be used, such as amino acids 2-12, aminoacids 3-14, 4-16, etc.

In each of the above-recited methods, α1-antitrypsin or analogs thereofare contemplated for use in a composition herein. These analogs mayinclude peptides. The peptides may include but are not limited to aminoacid peptides containing MPSSVSWGIL (SEQ. ID NO. 19); LAGLCCLVPV (SEQ.ID NO. 20) SLAEDPQGDA (SEQ. ID NO. 21); AQKTDTSHHD (SEQ. ID NO. 22)QDHPTFNKIT (SEQ. ID NO. 23); PNLAEFAFSL (SEQ. ID NO. 24); YRQLAHQSNS(SEQ. ID NO. 25); TNIFFSPVSI (SEQ. ID NO. 26); ATAFAMLSLG (SEQ. ID NO.27); TKADTHDEIL (SEQ. ID NO. 28); EGLNFNLTEI (SEQ. ID NO. 29);PEAQIHEGFQ (SEQ. ID) NO. 30); ELLRTLNQPD (SEQ. ID NO. 31); SQLQLTTGNG(SEQ. ID NO. 32); LFLSEGLKLV (SEQ. ID NO. 33); DKFLEDVKKL (SEQ. ID NO.34); YHSEAFTVNF (SEQ. ID NO. 35); GDHEEAKKQI (SEQ. ID NO. 36);NDYVEKGTQG (SEQ. ID NO. 37); KIVDLVKELD (SEQ. ID NO. 38); RDTVFALVNY(SEQ. 1D NO. 39); IFFKGKWERP (SEQ. ID NO. 40); FEVKDTEDED (SEQ. ID NO.41); FHVDQVTTVK (SEQ. ID NO. 42); VPMMKRLGMF (SEQ. ID NO. 43);NIQHCKKLSS (SEQ. ID NO. 44); WVLLMKYLGN (SEQ. ID NO. 45); ATAIFFLPDE(SEQ. ID NO. 46); GKLQHLENEL (SEQ. ID NO. 47); THDIITKFLE (SEQ. ED NO.48); NEDRRSASLH (SEQ. ID NO. 49); LPKLSITGTY (SEQ. ID NO. 50);DLKSVLGQLG (SEQ. ID NO. 51); ITKVFSNGAD (SEQ. ID NO. 52); LSGVTEEAPL(SEQ. ID NO. 53); KLSKAVHKAV (SEQ. ID NO. 54); LTIDEKGTEA (SEQ. ID NO.55); AGAMFLEAIP (SEQ. ID NO. 56); MSIPPEVKFN (SEQ. ID NO. 57);KPFVFLMIEQ (SEQ. ID NO. 58); NTKSPLFMGK (SEQ. ID NO. 59); VVNPTQK (SEQ.ID NO. 60), or any combination thereof

In Accordance with embodiments of the present invention, the peptide canbe protected or derivitized in by any means known in the art forexample, N-terminal acylation, C-terminal amidation, cyclization, etc.In a specific embodiment, the N-terminus of the peptide is acetylated.

Pharmaceutical Compositions

Embodiments herein provide for administration of compositions tosubjects in a biologically compatible form suitable for pharmaceuticaladministration in vivo. By “biologically compatible form suitable foradministration in vivo” is meant a form of the active agent (i.e.pharmaceutical chemical, protein, gene, antibody etc. of theembodiments) to be administered in which any toxic effects areoutweighed by the therapeutic effects of the active agent.Administration of a therapeutically active amount of the therapeuticcompositions is defined as an amount effective, at dosages and forperiods of time necessary to achieve the desired result. For example, atherapeutically active amount of a compound may vary according tofactors such as the disease state, age, sex, and weight of theindividual, and the ability of antibody to elicit a desired response inthe individual. Dosage regima may be adjusted to provide the optimumtherapeutic response.

In one embodiment, the compound (i.e. pharmaceutical chemical, protein,peptide etc. of the embodiments) may be administered in a convenientmanner such as subcutaneous, intravenous, by oral administration,inhalation, transdermal application, intravaginal application, topicalapplication, intranasal or rectal administration. Depending on the routeof administration, the active compound may be coated in a material toprotect the compound from the degradation by enzymes, acids and othernatural conditions that may inactivate the compound. In a preferredembodiment, the compound may be orally administered. In anotherpreferred embodiment, the compound may be administered intravenously. Inone particular embodiment, the compound may be administeredintranasally, such as inhalation.

A compound may be administered to a subject in an appropriate carrier ordiluent, co-administered with enzyme inhibitors or in an appropriatecarrier such as liposomes. The term “pharmaceutically acceptablecarrier” as used herein is intended to include diluents such as salineand aqueous buffer solutions. It may be necessary to coat the compoundwith, or co-administer the compound with, a material to prevent itsinactivation. The active agent may also be administered parenterally orintraperitoneally. Dispersions can also be prepared in glycerol, liquidpolyethylene glycols, and mixtures thereof and in oils. Under ordinaryconditions of storage and use, these preparations may contain apreservative to prevent the growth of microorganisms.

Pharmaceutical compositions suitable for injectable use may beadministered by means known in the art. For example, sterile aqueoussolutions (where water soluble) or dispersions and sterile powders forthe extemporaneous preparation of sterile injectable solutions ordispersion may be used. In all cases, the composition cant be sterileand can be fluid to the extent that easy syringability exists. It mightbe stable under the conditions of manufacture and storage and may bepreserved against the contaminating action of microorganisms such asbacteria and fungi. The pharmaceutically acceptable carrier can be asolvent or dispersion medium containing, for example, water, ethanol,polyol (for example, glycerol, propylene glycol, and liquidpolyetheylene glycol, and the like), and suitable mixtures thereof. Theproper fluidity can be maintained, for example, by the use of a coatingsuch as lecithin, by the maintenance of the required particle size inthe case of dispersion and by the use of surfactants. Prevention ofmicroorganisms can be achieved by heating, exposing the agent todetergent, irradiation or adding various antibacterial or antifungalagents.

Sterile injectable solutions can be prepared by incorporating activecompound (e.g. a compound that reduces serine protease activity) in therequired amount in an appropriate solvent with one or a combination ofingredients enumerated above, as required, followed by filteredsterilization.

Aqueous compositions can include an effective amount of a therapeuticcompound, peptide, epitopic core region, stimulator, inhibitor, and thelike, dissolved or dispersed in a pharmaceutically acceptable carrier oraqueous medium. Compounds and biological materials disclosed herein canbe purified by means known in the art.

Solutions of the active compounds as free-base or pharmacologicallyacceptable salts can be prepared in water suitably mixed with asurfactant, such as hydroxypropylcellulose. Dispersions can also beprepared in glycerol, liquid polyethylene glycols, and mixtures thereofand in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms. Prolonged absorption of the injectable compositions canbe brought about by the use in the compositions of agents delayingabsorption, for example, aluminum monostearate and gelatin.

Upon formulation, solutions will be administered in a manner compatiblewith the dosage formulation and in such amount as is therapeuticallyeffective. The formulations are easily administered in a variety ofdosage forms, such as the type of injectable solutions described above.It is contemplated that slow release capsules, timed-releasemicroparticles, and the like can also be employed. These particularaqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous and intraperitoneal administration.

The active therapeutic agents may be formulated within a mixture tocomprise about 0.0001 to 1.0 milligrams, or about 0.001 to 0.1milligrams, or about 0.1 to 1.0 or even about 1 to 10 gram per dose.Single dose or multiple doses can also be administered on an appropriateschedule for a predetermined condition.

In another embodiment, nasal solutions or sprays, aerosols or inhalantsmay be used to deliver the compound of interest. Additional formulationsthat are suitable for other modes of administration includesuppositories and pessaries. A rectal pessary or suppository may also beused. In general, for suppositories, traditional binders and carriersmay include, for example, polyalkylene glycols or triglycerides; suchsuppositories may be formed from mixtures containing the activeingredient in the range of 0.5% to 10%, preferably 1%-2%.

Oral formulations include such normally employed excipients as, forexample, pharmaceutical grades of mannitol, lactose, starch, magnesiumstearate, sodium saccharine, cellulose, magnesium carbonate and thelike. In certain defined embodiments, oral pharmaceutical compositionswill comprise an inert diluent or assimilable edible carrier, or theymay be enclosed in hard or soft shell gelatin capsule, or they may becompressed into tablets, or they may be incorporated directly with thefood of the diet. For oral therapeutic administration, the activecompounds may be incorporated with excipients and used in the form ofingestible tablets, buccal tables, troches, capsules, elixirs,suspensions, syrups, wafers, and the like. Such compositions andpreparations should contain at least 0.1% of active compound. Thepercentage of the compositions and preparations may, of course, bevaried and may conveniently be between about 2 to about 75% of theweight of the unit, or preferably between 25-60%. The amount of activecompounds in such therapeutically useful compositions is such that asuitable dosage will be obtained.

A pharmaceutical composition may be prepared with carriers that protectactive ingredients against rapid elimination from the body, such astime-release formulations or coatings. Such carriers include controlledrelease formulations, such as, but not limited to, microencapsulateddelivery systems, and biodegradable, biocompatible polymers, such asethylene vinyl acetate, polyanhydrides, polyglycolic acid,polyorthoesters, polylactic acid and others are known.

Pharmaceutical compositions are administered in an amount, and with afrequency, that is effective to inhibit or alleviate side effects of atransplant and/or to reduce or prevent rejection. The precise dosage andduration of treatment may be determined empirically using known testingprotocols or by testing the compositions in model systems known in theart and extrapolating therefrom. Dosages may also vary with the severityof the condition. A pharmaceutical composition is generally formulatedand administered to exert a therapeutically useful effect whileminimizing undesirable side effects. In general, an oral dose rangesfrom about 200 mg to about 1000 mg, which may be administered forexample 1 to 3 times per day.

It will be apparent that, for any particular subject, specific dosageregimens may be adjusted over time according to the individual need. Thepreferred doses for administration can be anywhere in a range betweenabout 0.01 mg and about 100 mg per ml of biologic fluid of treatedpatient. In one particular embodiment, the range can be between 1 and100 mg/kg which can be administered daily, every other day, biweekly,weekly, monthly etc. In another particular embodiment, the range can bebetween 10 and 75 mg/kg introduced weekly to a subject. Thetherapeutically effective amount of α1-antitrypsin, peptides, or drugsthat have similar activities as α1-antitrypsin or peptides can be alsomeasured in molar concentrations and can range between about 1 nM toabout 2 mM.

The tablets, troches, pills, capsules and the like may also contain thefollowing: a binder, as gum tragacanth, acacia, cornstarch, or gelatin;excipients, such as dicalcium phosphate; a disintegrating agent, such ascorn starch, potato starch, alginic acid and the like; a lubricant, suchas magnesium stearate; and a sweetening agent, such as sucrose, lactoseor saccharin may be added or a flavoring agent.

Liposomes can be used as a therapeutic delivery system and can beprepared in accordance with known laboratory techniques. In addition,dried lipids or lyophilized liposomes prepared as previously describedmay be reconstituted in a solution of active agent (e.g. nucleic acid,peptide, protein or chemical agent), and the solution diluted to anappropriate concentration with a suitable solvent known to those skilledin the art. The amount of active agent encapsulated can be determined inaccordance with standard methods.

In a preferred embodiment, a nucleic acid (e.g. α1-antitrypsin oranalogs thereof) and the lipid dioleoylphosphatidylcholine may beemployed. For example, nuclease-resistant oligonucleotides may be mixedwith lipids in the presence of excess t-butanol to generateliposomal-oligonucleotides for administration.

The pharmaceutical compositions containing the α1-antitrypsin, analogthereof, or inhibitor of serine protease activity or a functionalderivative thereof may be administered to individuals, particularlyhumans, for example by subcutaneously, intramuscularly, intranasally,orally, topically, transdermally, parenterally, gastrointestinally,transbronchially and transalveolarly. Topical administration isaccomplished via a topically applied cream, gel, rinse, etc. containingtherapeutically effective amounts of inhibitors of serine proteases.Transdermal administration is accomplished by application of a cream,rinse, gel, etc. capable of allowing the inhibitors of serine proteasesto penetrate the skin and enter the blood stream. In addition, osmoticpumps may be used for administration. The necessary dosage will varywith the particular condition being treated, method of administrationand rate of clearance of the molecule from the body.

In each of the aforementioned compositions and methods, a compoundhaving serine protease inhibitor activity and/or having α1-antitrypsinactivity or analog thereof may be used in a single therapeutic dose,acute manner or a chronic manner to treat episodes or prolonged bouts,respectively, in promoting graft survival, treating graft rejectionand/or associated graft rejection-induced side-effects.

In certain embodiments of the methods of the present invention, thesubject may be a mammal such as a human or a veterinary and/or adomesticated animal.

Therapeutic Methods

In one embodiment of the present invention, methods provide for treatinga subject in need of or undergoing a transplant. For example, treatmentsfor reducing graft rejection, promoting graft survival, and promotingprolonged graft function by administering to a subject in need thereof atherapeutically effective amount of a composition. The composition caninclude a compound capable of inhibiting at least one serine proteasefor example, α1-antitrypsin, or analog thereof.

Preserving the Graft During Transplant before Engraftment

According to the methods of the present invention, transplantationcomplications can be reduced or inhibited to obtain importanttherapeutic benefits. Therefore, administration of a therapeuticcomposition contemplated by embodiments of the invention, i.e.,α1-antitrypsin, derivative or analog thereof, can be beneficial for thetreatment of transplantation complications or conditions.

Another beneficial effect of use of the compositions and methods of thepresent invention include reducing negative effects on an organ ornon-organ during explant, isolation, transport and/or prior toimplantation. For example, the composition can reduce apoptosis, reduceproduction of cytokines, reduce production of NO, or combination thereofin an organ for transplant. In one particular embodiment, a compositioncan include a compound that includes alpha-1-antitrypsin, an analogthereof, a serine protease inhibitor, serine protease inhibitor-likeactivity, analog thereof or a combination thereof. The transplant organor non-organ can include but is not limited to, lung, kidney, heart,liver, soft tissue, skin, pancreas, intestine, soft tissue cornea, bonemarrow, stem cell, pancreatic islet, and combination thereof.

In a further embodiment, the methods and compositions of the inventionare useful in the therapeutic treatment of graft rejection associatedside effects. In a yet further embodiment, graft rejection associatedside effects can be prevented by the timely administration of the agentof the invention as a prophylactic, prior to onset of one or moresymptoms, or one or more signs, or prior to onset of one or more severesymptoms or one or more signs of a graft rejection associated disease.Thus, a patient at risk for a particular graft rejection or graftrejection-associated disease or clinical indication can be treated withserine protease inhibitors, for example,(Benzyloxycarbonyl)-L-Valyl-N-[1-(3-(5-(3-Trifluoromethylbenzyl)-1,2,4-oxadiazoly0carbonyl)-2-(S)-Methylpropyl]-L-Prolinamide; as a prophylactic measure.

It is contemplated herein that the present compositions and methods ofthe present invention can be used to treat patients with one or moregrafts who require chronic therapy to maintain graft integrity, and suchpatients will therefore benefit from indefinite or chronic use of therejection repressive therapy of the methods of the present invention.Yet another embodiment can be used to treat flairs of acute rejection soas to minimize the effects of acute clinical rejection, organ failure,and/or eventual destruction of the graft.

Desirable blood levels may be maintained by continuous infusion toprovide about 0.01-5.0 mg/kg/hr or by intermittent infusions containingabout 0.4-20 mg/kg of the active ingredient(s). Buffers, preservatives,antioxidants and the like can be incorporated as required. It isintended herein that the ranges recited also include all those specificpercentage amounts between the recited range. For example, the range ofabout 0.4 to 20 mg/kg also encompasses 0.5 to 19.9%, 0.6 to 19.8%, etc.,without actually reciting each specific range therewith,

Serine Protease Inhibitors

It is to be understood that the present invention is not limited to theexamples described herein, and other serine proteases known in the artcan be used within the limitations of the invention. For example, oneskilled in the art can easily adopt inhibitors as described in WO98/24806, which discloses substituted oxadiazole, thiadiazole andtriazole as serine protease inhibitors. U.S. Pat. No. 5,874,585discloses substituted heterocyclic compounds useful as inhibitors ofserine proteases for example,(benzyloxycarbonyl)-L-valyl-N41-(3-(5-(3-trifluoromethylbenzyl)-1,2,4-oxadiazolyl)carbonyl)-2-(S)-methylpropyll-L-prolinamidebenzyloxycarbonyl)-L-valyl-N-[1-(3-(5-(2-phenylethyl)-1,2,4-oxadiazolyl)carbonyl)-2-(S)-methylpropyl]-L-prolinamide;and(benzyloxycarbonyl)-L-valyl-N-[1-(3-(5-(2-methoxybenzyl)-1,2,4-oxadiazolyl)carbonyl)-2-(S)-methylpropyl]-L-prolinamide.

α1-antitrypsin is a glycoprotein of MW 51,000 with 417 amino acids and 3oligosaccharide side chains. Human α1-antitrypsin is a singlepolypeptide chain with no internal disulfide bonds and only a singlecysteine residue normally intermolecularly disulfide-linked to eithercysteine or glutathione. The reactive site of α1-antitrypsin contains amethionine residue, which is labile to oxidation upon exposure totobacco smoke or other oxidizing pollutants. Such oxidation reduces theelastase-inhibiting activity of α1-antitrypsin; therefore substitutionof another amino acid at that position, i.e. alanine, valine, glycine,phenylalanine, arginine or lysine, produces a form of α1-antitrypsinwhich is more stable. α1-antitrypsin can be represented by the followingformula:

SEQ ID 61: MPSSVSWGIL LAGLCCLVPV SLAEDPQGDA AQKTDTSHHD 100QDHPTFNKITPNLAEFAFSL YRQLAHQSNS TNIFFSPVSI  ATAFAMLSLG TKADTHDEILEGLNFNLTEI PEAQIHEGFQ ELLRTLNQPD SQLQLTTGNG 200LFLSEGLKLVDKFLEDVKKL YHSEAFTVNF GDHEEAKKQI  NDYVEKGTQG KIVDLVKELDRDTVFALVNY IFFKGKWERP FEVKDTEDED FHVDQVTTVK  300VPMMKRLGMF NIQHCKKLSS WVLLMKYLGN ATAIFFLPDE  GKLQHLENEL THDIITKFLENEDRRSASLH LPKLSITGTY DLKSVLGQLG ITKVFSNGAD  400LSGVTEEAPL KLSKAVHKAV LTIDEKGTEA AGAMFLEAIP  MSIPPEVKFN KP FVFLM IEQNTKSPLFMGK VVNPTQK 417

One important amino acid sequence near the carboxyterminal end ofα1-antitrypsin is shown in bold and underlined and is pertinent to thisinvention (details of the sequence can be found for example in U.S. Pat.No. 5,470,970, as incorporated by reference).

Extrahepatic sites of AAT production include neutrophils, monocytes andmacrophages, and the expression of AAT is inducible in response to LPS,TNFα, IL-1 and IL-6 in various cell types. Deficiency in AAT isassociated with immune dysfunctional conditions such as rheumatoidarthritis and systemic lupus erythematosus.

Other serine protease inhibitor molecules, which may be used in any ofthe disclosed compositions may include compounds disclosed in thefollowing: WO 98/20034 disclosing serine protease inhibitors from fleas;WO98/23565 disclosing aminoguanidine and alkoxyguanidine compoundsuseful for inhibiting serine proteases; WO98/50342 disclosingbis-aminomethylcarbonyl compounds useful for treating cysteine andserine protease disorders; WO98/50420 cyclic and other amino acidderivatives useful for thrombin-related diseases; WO 97/21690 disclosingD-amino acid containing derivatives; WO 97/10231 disclosingketomethylene group-containing inhibitors of serine and cysteineproteases; WO 97/03679 disclosing phosphorous containing inhibitors ofserine and cysteine proteases; WO 98/21186 benzothiazo and relatedheterocyclic inhibitors of serine proteases; WO 98/22619 disclosing acombination of inhibitors binding to P site of serine proteases withchelating site of divalent cations; WO 98/22098 disclosing a compositionwhich inhibits conversion of pro-enzyme CPP32 subfamily includingcaspase 3 (CPP32/Yama/Apopain); WO 97/48706 disclosingpyrrolo-pyrazine-diones; and WO 97/33996 disclosing human placentalbikunin (recombinant) as serine protease inhibitor.

Other compounds having serine protease inhibitory activity are equallysuitable and effective for use in the methods of the present invention,including but not limited to: tetrazole derivatives as disclosed in WO97/24339; guanidinobenzoic acid derivatives as disclosed in WO 97/37969and in a number of U.S. Pat. Nos. 4,283,418; 4,843,094; 4,310,533;4,283,418; 4,224,342; 4,021,472; 5,376,655; 5,247,084; and 5,077,428;phenylsulfonylamide derivatives represented by general formula in WO97/45402; novel sulfide, sulfoxide and sulfone derivatives representedby general formula in WO 97/49679; novel amidino derivatives representedby general formula in WO 99/41231; other amidinophenol derivatives asdisclosed in U.S. Pat. Nos. 5,432,178; 5,622,984; 5,614,555; 5,514,713;5,110,602; 5,004,612; and 4,889,723 among many others.

Graft Rejection and Graft Survival- Side- Effects and Conditions

One of the beneficial effects of use of the compositions and methods ofthe present invention include, for example, and not by way oflimitation, reduced infiltration of graft with cells or serum factors(including but not limited to, complement, anti graft antibody thatgenerate inflammation and graft rejection), reduced cytokines, reducednitric oxide, reduced apoptosis, and reduced specific immune responseagainst the graft or any combination thereof.

Management of Graft Rejection

By preventing or reducing the side effects or conditions associated withgraft survival or graft rejection using this novel approach, severaladvantages are obtained compared to alternative approaches, for example,and not by way of limitation:

1. Reduced infiltration of graft with cells or serum factors (forexample, and not by way of limitation, complement, anti graft antibodythat generate inflammation and graft rejection); reduced production ofcytokines or nitric oxide (NO) that can induce inflammation orapoptosis; inhibits apoptosis; inhibits immune activation, inhibits CMVor any combination thereof

2. Synthetic inhibitors of serine proteases (AAT-like mimics or analogs)can and have been developed by means known in the art. Such apharmaceutical agent may be formulated as for example, a cream to treatgraft rejection and/or promote graft survival.

3. Commercially available agents already approved for different use inhumans will work as a treatment for graft rejection and/or promote graftsurvival. These agents are currently used for indications other thangraft rejection and/or to promote graft survival, and include injectibleAAT, plasma preparations, aprotinin and others (American J. of RespCritical Care Med 1998, VII 158: 49-59, incorporated herein by referencein its entirety). In one embodiment, serine protease inhibitors may bedelivered by inhalation. An inhaled agent (natural AAT or a syntheticAAT-like mimic/or other inhibitor of serine protease) may be especiallyuseful due to elevated local concentrations, ease of drug delivery, andlack of side effects (since administration is not systemic). This modeof focused drug delivery may augment serine protease inhibitor activitywithin the lung tissues and associated lymphatics, which are two of theprincipal sites where diseases and/or clinical conditions associatedwith graft rejection and/or promotion of graft survival develop.

4. By promoting graft survival and/or treating graft rejection, thedirect cause of the side effect is disrupted in affected individuals.This invention specifically contemplates inhibiting host cell serineproteases or induce the SEC receptor or combination thereof as a methodof treating graft rejection and/or promoting graft survival in a mammalin need thereof in conjunction with administration of one or moreanti-rejection and/or anti-microbial.

5. There is an extensive clinical experience using injectible AAT totreat patients with genetic AAT deficiency. No long-term negativeeffects have been detected to date (American J. of Resp Critical CareMed 1998, VII 158: 49-59; Wencker et al. Chest 2001 119:737-744).Moreover, a small molecule inhibitor of host serine protease has beenadministered to patients with Kawasaki's Disease (Ulinistatin, Onopharmaceuticals).

Isolated Proteins For Use In The Compositions And Methods Of TheInvention

One aspect of the invention pertains to proteins, and portions thereof,as well as polypeptide fragments suitable for use as immunogens to raiseantibodies directed against a polypeptide of the invention. In oneembodiment, the native polypeptide can be isolated from cells or tissuesources by an appropriate purification scheme using standard proteinpurification techniques. In another embodiment, polypeptides of theinvention are produced by recombinant DNA techniques. Alternative torecombinant expression, a polypeptide of the invention can besynthesized chemically using standard peptide synthesis techniques.

Recombinant unmodified and mutant variants of .alpha.sub.1-antitrypsinproduced by genetic engineering methods are also known (see U.S. Pat.No. 4,711,848). The nucleotide sequence of human alpha.sub.1-antitrypsinand other human alpha.sub.1-antitrypsin variants has been disclosed ininternational published application No. WO 86/00,337, the entirecontents of which are incorporated herein by reference. This nucleotidesequence may be used as starting material to generate all of the AATamino acid variants and amino acid fragments depicted herein, usingrecombinant DNA techniques and methods known to those of skill in theart.

An isolated and/or purified or partially purified protein orbiologically active portion thereof may be used in any embodiment of theinvention. A protein that is substantially free of cellular materialincludes preparations of protein having less than about 30%, 20%, 10%,or 5% (by dry weight) of heterologous protein. When the protein orbiologically active portion thereof is recombinantly produced, it canalso be substantially free of culture medium. When the protein isproduced by chemical synthesis, it is preferably substantially free ofchemical precursors or other chemicals. Accordingly, such preparationsof the protein have less than about 30%, 20%, 10%, and 5% (by dryweight) of chemical precursors or compounds other than the polypeptideof interest.

Biologically active portions of a polypeptide of the invention includepolypeptides including amino acid sequences sufficiently identical to orderived from the amino acid sequence of the protein (e.g., the aminoacid sequence shown in any of SEQ ID Nos: 1 to 60, which exhibit atleast one activity of the corresponding full-length protein). Abiologically active portion of a protein of the invention can be apolypeptide, which is, for example, 5, 10, 25, 50, 100 or more aminoacids in length. Moreover, other biologically active portions, in whichother regions of the protein are deleted, can be prepared by recombinanttechniques and evaluated for one or more of the functional activities ofthe native form of a polypeptide of the invention.

Preferred polypeptides have the amino acid sequence of SEQ ID Nos: 1 to60. Other useful proteins are substantially identical (e.g., at leastabout 45%, preferably 55%, 65%, 75%, 85%, 95%, or 99%) to any of SEQ IDNOs: 1 to 60, and retain the functional activity of the protein of thecorresponding naturally-occurring protein yet differ in amino acidsequence due to natural allelic variation or mutagenesis.

The compounds of the present invention can be used as therapeutic agentsin the treatment of a physiological (especially pathological) conditioncaused in whole or part, by excessive serine protease activity. Inaddition, a physiological (especially pathological) condition can beinhibited in whole or part. Peptides contemplated herein may beadministered as free peptides or pharmaceutically acceptable saltsthereof The peptides should be administered to individuals as apharmaceutical composition, which, in most cases, will include thepeptide and/or pharmaceutical salts thereof with a pharmaceuticallyacceptable carrier.

When utilizing BLAST, Gapped BLAST, and PSI-Blast programs, the defaultparameters of the respective programs (e.g., XBLAST and NBLAST) can beused. See http://www.ncbi.nlm.nih.gov.

The present invention also pertains to variants of the polypeptides ofthe invention. Such variants have an altered amino acid sequence whichcan function as either agonists (mimetics) or as antagonists. Variantscan be generated by mutagenesis, e.g., discrete point mutation ortruncation. An agonist can retain substantially the same, or a subset,of the biological activities of the naturally occurring form of theprotein. An antagonist of a protein can inhibit one or more of theactivities of the naturally occurring form of the protein by, forexample, competitively binding to a downstream or upstream member of acellular signaling cascade which includes the protein of interest. Thus,specific biological effects can be elicited by treatment with a variantof limited function. Treatment of a subject with a variant having asubset of the biological activities of the naturally occurring form ofthe protein can have fewer side effects in a subject relative totreatment with the naturally occurring form of the protein.

Variants of a protein of the invention which function as either agonists(mimetics) or as antagonists can be identified by screeningcombinatorial libraries of mutants, e.g., truncation mutants, of theprotein of the invention for agonist or antagonist activity.

Fusion Polypeptides

In other embodiments, compounds having serine protease inhibitoractivity such as α1-antitrypsin and/or analog thereof, may be part of afusion polypeptide. In one example, a fusion polypeptide may includeα1-antitrypsin (e.g. mammalian α1-antitrypsin) or an analog thereof anda different amino acid sequence that may be heterologous to theα1-antitrypsin or analog substance.

In yet other embodiments, the fusion polypeptide contemplated for use inthe methods of the present invention can additionally include an aminoacid sequence that is useful for identifying, tracking or purifying thefusion polypeptide, e.g., a FLAG or HIS tag sequence. The fusionpolypeptide can include a proteolytic cleavage site that can remove theheterologous amino acid sequence from the compound capable of serineprotease inhibition, such as mammalian α1-antitrypsin or analog thereof

In one embodiment, fusion polypeptides of the invention are produced byrecombinant DNA techniques. Alternative to recombinant expression, afusion polypeptide of the invention can be synthesized chemically usingstandard peptide synthesis techniques. The present invention alsoprovides compositions that comprise a fusion polypeptide of theinvention and a pharmaceutically acceptable carrier, excipient ordiluent.

In particular, in one embodiment the fusion protein comprises aheterologous sequence that is a sequence derived from a member of theimmunoglobulin protein family, for example, comprise an immunoglobulinconstant region, e.g., a human immunoglobulin constant region such as ahuman IgG1 constant region. The fusion protein can, for example, includea portion of α1-antitrypsin, analog thereof or inhibitor of serineprotease activity polypeptide fused with the amino-terminus or thecarboxyl-terminus of an immunoglobulin constant region, as disclosed,e.g., in U.S. Pat. No. 5,714,147, and U.S. Pat. No. 5,116,964. Inaccordance with these embodiments, the FcR region of the immunoglobulinmay be either wild-type or mutated. In certain embodiments, it isdesirable to utilize an immunoglobulin fusion protein that does notinteract with an Fc receptor and does not initiate ADCC reactions. Insuch instances, the immunoglobulin heterologous sequence of the fusionprotein can be mutated to inhibit such reactions. See, e.g., U.S. Pat.No. 5,985,279 and WO 98/06248.

In yet another embodiment, α1-antitrypsin, analog thereof, or inhibitorof serine protease activity polypeptide fusion protein comprises a GSTfusion protein in which is fused to the C-terminus of GST sequences.Fusion expression vectors and purification and detection means are knownin the art.

Expression vectors can routinely be designed for expression of a fusionpolypeptide of the invention in prokaryotic (e.g., E. coil) oreukaryotic cells (e.g., insect cells (using baculovirus expressionvectors), yeast cells or mammalian cells) by means known in the art.

Expression of proteins in prokaryotes may be carried out by means knownin the art. Such fusion vectors typically serve three purposes: 1) toincrease expression of recombinant protein; 2) to increase thesolubility of the recombinant protein; and 3) to aid in the purificationof the recombinant protein by acting as a ligand in affinitypurification.

In yet another embodiment, a nucleic acid of the invention is expressedin mammalian cells using a mammalian expression vector as described inthe art. In another embodiment, the recombinant mammalian expressionvector is capable of directing expression of the nucleic acidpreferentially in a particular cell type (e.g., tissue-specificregulatory elements are used to express the nucleic acid) such aspancreas-specific promoters (Edlund et al. (1985) Science 230:912-916),and mammary gland-specific promoters (e.g., milk whey promoter; U.S.Pat. No. 4,873,316 and European Application Publication No. 264,166). Ahost cell can be any prokaryotic (e.g., E. coli) or eukaryotic cell(e.g., insect cells, yeast or mammalian cells). Vector DNA can beintroduced into prokaryotic or eukaryotic cells via conventionaltransformation or transfection techniques.

Combination Therapies

In each of the aforementioned methods of the present invention, the useof a compound capable of inhibiting serine protease or α1-antitrypsin oranalog thereof alone or in combination with standard immunosuppressiveagents enables transplantation of grafts into immunosuppressed orimmunocompromised recipients. This combination therapy will expand theeligible patient population able to receive this form of treatment.

In each of the aforementioned aspects and embodiments of the invention,combination therapies other than those already enumerated above are alsospecifically contemplated herein. In particular, the compositions of thepresent invention may be admininistered with one or more macrolide ornon-macrolide antibiotics, anti-bacterial agents, anti-fungals,anti-viral agents, and anti-parasitic agents. Examples of macrolideantibiotics that may be used in combination with the composition of thepresent invention include but are not limited to synthetic,semi-synthetic or naturally occurring macrolidic antibiotic compounds:methymycin, neomethymycin, YC-17, litorin, TMP-SSX, erythromycin A to F,and oleandomycin. Examples of preferred erythromycin anderythromycin-like compounds include: erythromycin, clarithromycin,azithromycin, and troleandomycin.

Examples of anti-bacterial agents include, but are not limited to,penicillins, quinolonses, aminoglycosides, vancomycin, monobactams,cephalosporins, carbacephems, cephamycins, carbapenems, and monobactamsand their various salts, acids, bases, and other derivatives.

Anti-fungal agents include, but are not limited to, caspofungin,terbinafine hydrochloride, nystatin, and selenium sulfide.

Anti-viral agents include, but are not limited to, gancyclovir,acyclovir, valacylocir, amantadine hydrochloride, rimantadin andedoxudine

Examples of macrolide antibiotics that may be used in combination withthe composition of the present invention include but are not limited tosynthetic, semi-synthetic or naturally occurring macrolidic antibioticcompounds: methymycin, neomethymycin, YC-17, litorin, TMP-SSX,erythromycin A to F, and oleandomycin. Examples of preferrederythromycin and erythromycin-like compounds include: erythromycin,clarithromycin, azithromycin, and troleandomycin.

Anti-parasitic agents include, but are not limited to,pirethrins/piperonyl butoxide, permethrin, iodoquinol, metronidazole,co-trimoxazole (sulfamethoxazole/trimethoprim), and pentamidineisethionate.

In another aspect, in the method of the present invention, one may, forexample, supplement the composition by administration of atherapeutically effective amount of one or more an anti-inflammatory orimmunomodulatory drugs or agents. By “anti-inflammatory drugs”, it ismeant, e.g., agents which treat inflammatory responses, i.e., a tissuereaction to injury, e.g., agents which treat the immune, vascular, orlymphatic systems.

Anti-inflammatory or immunomodulatory drugs or agents suitable for usein this invention include, but are not limited to, interferonderivatives, (e.g., betaseron); prostane derivatives, (e.g., compoundsdisclosed in PCT/DE93/0013, iloprost, cortisol, dexamethasone;immunsuppressives, (e.g., cyclosporine A, FK-506 (mycophenylatemofetil); lipoxygenase inhibitors, (e.g., zileutone, MK-886, WY-50295);leukotriene antagonists, (e.g., compounds disclosed in DE 40091171German patent application P 42 42 390.2); and analogs; peptidederivatives, (e.g., ACTH and analogs); soluble TNF-receptors;TNF-antibodies; soluble receptors of interleukins, other cytokines,T-cell-proteins; antibodies against receptors of interleukins, othercytokines, and T-cell-proteins.

Kits

In still further embodiments, the present invention concerns kits foruse with the methods described above. Small molecules, proteins orpeptides may be employed for use in any of the disclosed methods. Inaddition, other agents such as anti-bacterial agents, immunosuppressiveagents, anti-inflammatory agents may be provided in the kit. The kitswill thus can include, in suitable container means, a protein or apeptide or analog agent, and optionally one or more additional agents.

The kits may further include a suitably aliquoted composition of theencoded protein or polypeptide antigen, whether labeled or unlabeled, asmay be used to prepare a standard curve for a detection assay.

The container means of the kits will generally include at least onevial, test tube, flask, bottle, syringe or other container means, intowhich the antibody or antigen may be placed, and preferably, suitablyaliquoted. Where a second or third binding ligand or additionalcomponent is provided, the kit will also generally contain a second,third or other additional container into which this ligand or componentmay be placed. The kits of the present invention will also typicallyinclude a means for containing the antibody, antigen, and any otherreagent containers in close confinement for commercial sale. Suchcontainers may include injection or blow-molded plastic containers intowhich the desired vials are retained.

EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventors to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1

Alpha-1-antitrypsin Prolongs Graft Islet Graft Survival in Mice

FIG. 1A-1D. Islets from DBA/2 mice (H-2d) were transplanted under therenal capsule of streptozotocin-induced hyperglycemic C57BL/6 mice(H-2b). (A) Glucose levels from days 6-18. Control consists of mice thatwere untreated (n=3) or treated from day −1 every 3 days with humanalbumin (ALB, 6 mg, n=3). Prolonged islet graft survival is observed inmice treated from day −1 every 3 days with human AAT (2 mg, n=10).*P<0.05, **P<0.01, ***P<0.001 between glucose levels on same day. (B)Treatment protocols. Control and full AAT treatment are described inpanel A. Early AAT treatment consists of treatment on days −1, 1 and 3(2 mg, n=3). Late AAT treatment consists of treatment from day 2 and onevery 2 days (2 mg, n=3). Rejection indicates the day that glucoselevels exceed 300 mg/dl. (C) Effect of mouse anti-human-AAT antibodies.Dashed line indicates post transplantation glucose levels of a mouseunder full AAT treatment protocol (see A, B) that was immunized bymultiple administrations of human AAT prior to transplantation (1representative, n=3). Solid line indicates glucose levels of anon-immunized mouse treated under full AAT treatment protocol (1representative, n=10). Arrow indicates detection of treatment-induced,anti-human-AAT antibodies in the non-immunized representative mouse. (D)Comparison of day 15 post-transplantation glucose levels in mice thatwere under full treatment protocol with ALB (n=3) or AAT (non-immunizedn=10, immunized n=3). Of the AAT-treated group, antibodies were detectedon day 15 in 3/3 immunized mice and in 6/10 non-immunized mice.**P=0.005 between mice that produced antibodies (n=6) and mice that didnot produce antibodies (n=4).

Treatment with human albumin (6 mg) resulted in graft rejectioncomparable to that of untreated recipient mice. In contrast, recipientmice that received AAT (2 mg) exhibited prolonged graft function. Asdepicted in FIG. 1b , neither of the partial treatment protocols, i.e.,days −1, 1 and 3 (‘early treatment’) or days 2 and beyond (‘latetreatment’) prolonged allograft survival.

AAT-treated mice developed anti-human-AAT antibodies (FIGS. 1C and D).Individual mice exhibited anti-human-AAT antibodies at various timepoints (data not shown). To ascertain that the antibodies reduce theprotective effect of AAT, a group of mice was pre-exposed (“immunized”)to human AAT two months before being rendered hyperglycemic andtransplanted with allogeneic islets. These graft recipients were treatedwith the full AAT protocol, despite exhibiting high titers of specificantibodies before engraftment, and displayed rapid graft rejection (FIG.1C). Day 15 was chosen to depict an association between antibodyformation and loss of AAT protective activity; at this time pointAAT-treated mice were divided into positive and negative producers ofanti-human-AAT antibodies. As shown in FIG. 1D, on day 15 allantibody-positive mice were hyperglycemic and all antibody-negative micewere normoglycemic.

Example 2

FIG. 2A-2D illustrates an exemplary method of the effect of AAT onthioglycolate-elicited peritoneal cellular infiltrates. Mice wereadministered intraperitoneal 0.1 ml saline, ALB, AAT or oxidized-AATfollowed by 1 ml of saline or thioglycolate (ThG, 3% w/v, n=3 pergroup). Peritoneal lavage was performed on separate groups after 24 and48 hours. (A) Total cell population of lavaged cells of (open bars)saline or (closed bars) AAT-treated (5 mg) thioglycolate-injected mice.**P<0.05. (B) Percent cell population from saline-treated mice at 48hours. ** P<0.05. (C) Oxidation of AAT. AAT was subjected to oxidativeradicals (see Methods). Loss of serine protease activity of oxidized AATwas assessed in an elastase assay. Activity of elastase in the absenceof native AAT was set at 100% and the percentage of activity in thepresence of native and oxidized AAT was calculated (n=3). ***P<0.001. InFIG. 2D, elicited macrophages and neutrophils are identified. Peritonealinfiltrates from 48 hour lavages of ALB (6 mg) and AAT-treated (6 mg),thioglycolate-injected mice were stained for FACS analysis by specificantibodies. Macrophages and neutrophils were identified on the basis ofF4/80 and GR1 versus side scatter flow cytometry profiles. Top, FACSanalysis representative graphs (n=3). Quantified FACS results (n=3) aredepicted in the bottom.

AAT Inhibits Cellular Infiltration

To address the possibility that AAT affects effector cell infiltration,two models of cell emigration were examined: thioglycolate(ThG)-elicited peritoneal infiltration, and cellular infiltration due tointraperitoneal injection of MHC-incompatible fibroblasts.

As shown in FIG. 2A, there was a progressive increase in total cellcount at 24 and 48 hours in mice injected with ThG, whereas nosignificant increase was observed in mice injected with AAT and ThG. At48 hours, total cell count in peritoneal lavage of AAT-treated mice was50% of that of control (FIG. 2B). Total cell count in mice that receivedalbumin control was similar to that of saline-treated mice. There was adose-dependent effect of AAT in that one-sixth the dose was found toreduce cell count to a lesser extent in a significant manner. OxidizedAAT, which had lost its in vitro anti-elastase activity (FIG. 2C), didnot affect cellular infiltrate at 1 mg (FIG. 2B).

The decrease in total cell count is primarily attributed to a decreasein the number of neutrophils (FIG. 2D), identified by theirGR-1high/intermediate side-scatter (SSC) profile. No major differencewas observed with the infiltration of macrophages, identified by theirF4/80int, GR-1int, intermediate SSC profile¹², which is distinct fromthe F4/80very high, GR-1low, high SSC profile of resident macrophages¹²(data not shown).

Example 3

FIG. 3A-3C illustrates an exemplary method of the effect of AAT onMHC-incompatible, NIH-3T3-fibroblast-elicited peritoneal cellularinfiltrates. Mice (C57BL/6; H-2b) were injected i.p. 0.1 ml saline orAAT (1 mg) followed by 1 ml NIH-3T3 cells (1′107 cells in saline; H-2d).Peritoneal lavage was performed daily on days 1-5 and cellsubpopulations were identified by FACS analysis. (n=3 per treatment).(A) Cell numbers. The number of cells in each subpopulation wascalculated from the percentages obtained by FACS analysis, and totalnumber of cells in the infiltrate. *P<0.05, **P<0.01 between cellnumbers on the same day. (B) Representative FACS analysis. (C) Effect ofAAT on intensity and function of infiltrate elicited by islet allograft.Left, Hematoxilyn and Eosin (H&E) staining of day 7 islet allografts. Asection of AAT-treated islet graft (white frame) is compared to asimilar section of ALB-treated diabetic recipient mouse (full treatmentprotocol, see FIG. 1A). Arrow points at border between islet andsurrounding infiltrate. Right, Immunohistochemistry (IHC) withanti-insulin antibodies of day 15 islet grafts. A section of autologousislet graft (white frame) is compared to similar sections of allograftsof AAT- and ALB-treated recipient mice. R, renal parenchyma, G, graft,C, renal capsule.

As illustrated in FIG. 3A, introduction of allogeneic cells evoked acellular infiltrate that consisted of early appearing neutrophils andactivated macrophages, and late appearing CD3+ and NK cells (FIG. 3B).AAT-treated mice exhibited a reduction in neutrophils, CD3+ and NKcells, dark color is insulin staining.

To evaluate the level of cellular infiltration into grafted islets,grafts from AAT- and ALB-treated recipient mice were removed on day 7,fixed in paraformaldehyde and stained with Hematoxilin and Eosin. Asdepicted in FIG. 3C (left), a cellular infiltrate is demonstrableregardless of AAT treatment, and includes neutrophils and lymphocytes.However, the infiltrates evoked by grafts of ALB-treated recipient micewere more massive and cause the disruption of islet borders, compared tointact islets of AAT-treated recipient mice. To evaluate islet function,grafts from AAT- and ALB-treated recipient mice were removed on day 15,and immunohistochemistry was performed with anti-insulin antibodies,dark color is insulin staining. As depicted in FIG. 3C (right), insulinproduction is preserved on day 15 in islets of AAT-treated recipients.

Example 4

FIG. 4A-4H illustrates an exemplary method of the effect of AAT on isletresponses. (A-D) Islets from C57BL/6 mice were cultured at 100islets/well, in duplicate. AAT was incubated at the indicatedconcentrations for 1 hour before the addition of IFNγ (5 ng/ml) plusIL-1β (10 ng/ml). 72 hours later, supernatants were collected and isletviability was assessed. Islet cells responses in the absence of AAT wereset at 100%. Data are combined from 3 individual experiments, induplicate. **P<0.01, ***P<0.001 between AAT-treated and untreatedislets. Mean±SEM of a. nitrite levels, b. Cell viability and c. MIP-1αlevels. Dashed line represents islets incubated at one-30th theconcentration of IFNγ/IL-1β. d. TNFα levels. (E) Insulin inductionassay. Islets were incubated in triplicate (20 islets/well) in thepresence of AAT (0.5 mg/ml) or ALB (0.5 mg/ml) 1 hour before addition ofIFNγ (5 ng/ml) plus IL-1β (10 ng/ml). 24 hours later, islets weretransferred to a 3 mM or 20 mM glucose solution for 30 minutes andinsulin levels were measured. Vertical axis depicts the ratio betweeninsulin levels at both glucose concentrations. *P<0.05 betweenAAT-treated and ALB-treated islets. (F) Streptozotocin toxicity. C57BL/6mice were injected i.p. with AAT (5 mg) or saline, one day before, onsame day and one day after injection of streptozotocin (225 mg/kg) orsaline (n=3 per group). 48 hours later, pancreata were removed andinsulin-containing cells were identified by immunohistochemistry. Eachimage depicts a representative islet form one pancreas. Graph, mean±SEMpercent change of insulin-containing cells as determined manually fromimages of 2 islets per pancreas (n=6 per treatment group). *P<0.05. (G)Cellular content of islets. Freshly isolated islets (100 islets intriplicate) and residual non-islet pancreatic debris were dissociatedinto single cell suspensions and stained for FACS analysis withanti-CD45-APC or isotype control antibody. Shaded area, islets. Openarea, debris. (H) MHC class II expression. Islets from C57BL/6 mice werecultured (100 islets/well in duplicate) in the presence of AAT (0.5mg/ml) 1 hour before the addition of IFNγ (5 ng/ml) plus IL-1β (10ng/ml). 24 hours later, islets were dissociated into single cellsuspensions and double-stained for FACS analysis with anti-CD45-APC andanti-MHCII-PE, or isotype control antibodies. Left, Mean±SEM percentchange from control (CT) unstimulated islets. *P<0.05 betweenAAT-treated and untreated islets. Right, Representative FACS analysis;Shaded area, AAT-treated islets. Open area, stimulated islets. Eventsare gated for CD45+.

AAT Modifies Islet Response to Proinflammatory Mediators

Various islet responses to IL-1β/IFNγ were examined in vitro. Isletsexposed to IL-IL-1β/IFNγ for 72 hours produce nitric oxide (NO) in aconcentration-dependent manner and exhibit NO-dependent loss ofviability. As shown in FIGS. 4A and B, in the presence of AAT, less NOwas produced and greater islet viability was obtained. The production ofMIP-1α was decreased in the presence of AAT, particularly whenstimulated by low concentrations of IL-1β/IFNγ (FIG. 4C). Notably, TNFαlevel in supernatants was markedly diminished by AAT (FIG. 4D). Insulininduction was inhibited by IL-1β/IFNγ, but was intact in the presence ofIL-1β/IFNγ plus AAT (FIG. 4E). To test the effect of AAT on islets invivo, STZ toxicity was evaluated. AAT (2 mg) was administered one daybefore, on the same day and a day after STZ injection.Immunohistochemistry of pancreata with anti-insulin antibodies at 48hours after STZ injection reveals more insulin-producing cells in isletsof AAT- than ALB-treated mice (26.3%±2.6 and 12.8%±2.3 insulin-producingcells per islet, respectively, FIG. 4f ). White cell content of freshlyisolated islets was evaluated by FACS analysis. Islets contain CD45+cells (FIG. 4G) that are also positive for the monocytic/granulocyticmarkers GR1 and F4/80 (data not shown). This cell population respondedto AAT with decreased surface MHC class II (FIG. 4H).

Example 5

FIG. 5A-5D illustrates the effect of AAT on TNF-α. (A) Islets fromC57BL/6 mice were cultured (100 islets/well in triplicate) in thepresence of AAT (0.5 mg/ml) or TACE inhibitor (10 mM) 1 hour beforestimulation by IFNγ (5 ng/ml) plus IL-1β (10 ng/ml). Left, mean±SEMchange in TNFγ in supernatants after 72 hours of incubation. Right,mean±SEM fold change in membrane TNFα on islet cells after 5 hours ofincubation, according to FACS analysis. ***P<0.001 compared control (CT)levels in the absence of AAT. (B) Representative FACS analysis ofmembrane TNFα on stimulated islet cells in the absence (open area) orpresence (shaded area) of AAT. Events are gated for CD45+. (C)Streptozotocin-induced hyperglycemia. C57BL/6 mice were injected i.p.with saline (n=3), AAT (5 mg, n=3) or TNFα (1 mg/kg, n=3) oradministered p.o. with TACE inhibitor (TACEi, 60 mg/kg, n=6) one daybefore injection of STZ (225 mg/kg, i.p.). Subsequently, AAT and TNFαwere injected daily; TACE inhibitor was administered twice a day. At 48hours, mean±SEM glucose levels are compared to those of normallittermates (n=3). *P<0.05, **P<0.01 compared to saline-treated,STZ-injected mice.

AAT Inhibits Release of Membrane TNFα

Proteolytic cleavage of membrane TNFα releases soluble TNFα fromactivated cells by the action of TNFα-converting-enzyme (TACE). Theinventors examined the levels of membrane TNFα on stimulated islets inthe presence of AAT. The effect of AAT was compared to that of a TACEinhibitor. Both AAT and TACE inhibitor decreased TNFα levels insupernatants of islets exposed to IL-1β/IFNγ (FIG. 5A, left). Underthese conditions, membrane TNFα accumulated on the cell surface ofCD45+islet cells (FIG. 5A, right).

To assess the possibility that islet protection occurs via inhibition ofrelease of membrane TNFα in vivo, TACE inhibitor, p75 TNF receptor (TNFBP) or AAT were introduced to mice prior to STZ injection. Although allmice developed hyperglycemia after day 4, the progression of β-celltoxicity was significantly affected by treatments. As shown in FIG. □5C,the effect of STZ at 48 hours was decreased in the presence of AAT (adecrease of 23.2%±2.3 in fasting glucose levels compared to STZ/salineinjected mice). The effect of TACE inhibitor and p75 TNF receptor wasnot as profound. Similarly, TACE inhibitor prolonged islet graftsurvival to a lesser extent than AAT (preliminary data not shown).

Splenocytes that were harvested 48 hours after ThG injection producedTNFα in culture (FIG. 5D). AAT administered prior to thioglycolatedecreased TNFα release from cultured splenocytes. A similar trend wasfound with IFNγ (data not shown), signifying that the response to ThGhad effects that extend beyond the peritoneal compartment and thatpretreatment with AAT reduced these effects.

Example 6

FIG. 6A-6D illustrates the effect of AAT on Islet allografttransplantation. 6A illustrates the time course study aftertransplantation of islet cells. This example indicates that treated micemaintain normoglycemia over a 60 day period (n=4), after the AAT therapywas withdrawn. After withdraw of the therapy, the normoglycemia lastedanother 20 days. 6A illustrates the glucose follow-up. Positive insulinstaining in a day-85 treated islet graft was also demonstrated (data notshown). 6B illustrates an immune infiltrate found outside the graftarea. 6C illustrates an increase in the presence of CD4+ and acomparative decrease in monocytes and neutrophils. It was also shownthat massive vascularization was evident inside the graft (data notshown). It has been observed that long-lasting accepted islet grafts canbe spared from an immune alloresponse even after therapy removal,whether the therapy had evoked an immune tolerance specific for thestrain of donor islets was evaluated. For this, grafts were explanted bynephrectomy and the now-hyperglycemic original recipients werere-transplanted with either the same strain of islets as before (n=2),or a 3^(rd) strain which they had never encountered before (n=2). Inaccordance with established strain specific immune tolerance, miceaccepted grafts from original donors, but had acutely rejected3^(rd)-strain grafts (6D); the same donor (left) and a 3^(rd) donorre-graft (right).

Example 7

FIG. 7A-7E illustrates the production of AAT by islet cell andreflection of islet graft survival. 7A illustrates a time corseexpression of mouse AAT mRNA after cytokine production (IL-1β and IFNγ)(left) and at 8 hours (right). To demonstrate the relevance ofendogenous alpha-1-antitrypsin in physiological conditions, the issue ofislet injury during pancreatitis was addressed. In mouse model of acutepancreatitis, isolated islets of pancreata that are inflamed expressinducible alpha-1-antitrypsin. 7B illustrates an example of islet injuryduring pancreatitis; the histology of normal islets (top left), thehistology of islets of an inflamed pancreas (top right) and expressionof mouse AAT in islets obtained from the pancreata in an acutepancreatitis model (bottom). Alpha-1-antitrypsin levels duringpancreatitis (caerulein model for acute pancreatitis). Top, histology ofan islet in a normal pancreas (left) and an islet in an inflamedpancreas (right), representative of n=3. Bottom, expression of mousealpha-1-antitrypsin in islets obtained from pancreata in acutepancreatitis model. Treatment of mice with exogenous alpha-1-antitrypsinresulted in down-regulation of endogenous alpha-1-antitrypsinexpression, as well as decrease in serum TNFα levels (not shown).

To demonstrate the relevance of endogenous alpha-1-antitrypsin in islettransplantation, islet allografts from untreated transplanted mice ondays 1 through 7 after transplantation (n=3) were excised. These wereexamined for alpha-1-antitrypsin expression and reveal a pattern whichmay fit inflammation phase (days 1-3) followed by loss of islet mass(days 4-7). 7C illustrates an example of samples of islet allograftstaken post grafting and percent change in AAT mRNA levels were alsoassessed. Total RNA was extracted and mRNA for alpha-1-antitrypsinevaluated by RT-PCR.

Islet protection from cytokine injury was examined using endogenousalpha-1-antitrypsin by introducing oncostatin M, a member of IL-6 familythat induces alpha-1-antitrypsin expression in islets without causingislet death. After 4 days that human islets were incubated withoncostatin M, for the purpose of accumulation of sufficientalpha-1-antitrypsin, islets were added the β-cell-toxic combination ofIL-1β/IFNγ. Pretreated islets that had excess alpha-1-antitrypsin wereprotected from injury, supporting the concept that islet-derivedalpha-1-antitrypsin may participate in islet protection duringinflammation. 7D illustrates an example of islet protection fromcytokine injury with endogenous AAT by introducing oncostatin M (aninterleukin 6 (IL-6) family member) that induces AAT expression inislets, oncostatin M and AAT levels (top left); nitric oxide andviability levels assessed (top right). Bottom, human islets exposed tooncostatin M for 4 days produce enough alpha-1-antitrypsin to diminishthe effects of IL-1β/IFNγ added for an additional 48 hours.

Example 8

In one exemplary study, alpha-1-antitrypsin on human islets wasexamined. FIG. 8A-8D illustrates the effect of AAT on human islets. Theproduction of nitric oxide (8A), TNF-α production (8B) IL-6 (8C) andIL-8 (8D) was examined.100 human islets per well were seeded intriplicates and added alpha-1-antitrypsin (AAT) 2 hours before stimuli.Supernatants were assayed 72 hours later. 3A, nitric oxide; 3B, TNFα;3C, IL-6; 3D, IL-8. Results are mean ±SEM and are representative ofseparate islet isolations from three human donors.

METHODS

In accordance with the present invention there may be employedconventional molecular biology, microbiology, and recombinant DNAtechniques within the skill of the art. Such techniques are explainedfully in the literature. See, e.g., Sambrook, Fritsch & Maniatis,Molecular Cloning: A Laboratory Manual, Second Edition 1989, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y.; Animal Cell Culture,R. I. Freshney, ed., 1986).

Mice. C57BL/6 and DBA/2 females were purchased from JacksonLaboratories.

Induction of Hyperglycemia by Streptozotocin, Islet Isolation and IsletTransplantation.

In one exemplary method, 5-6 weeks old C57BL/6 mice were treatedintraperitoneally (i.p.) with 225 mg/kg Streptozotocin (STZ) (Sigma).Mice with established hyperglycemia were used at least 5 days after STZadministration. Islets were isolated from DBA/2 mice on day oftransplantation, or 24 hours before in vitro assays, by enzymaticdigestion of pancreata, by means known in the art, with minormodifications. Briefly, mice were anesthetized with i.p. ketamine (50mg/kg, Vedco Inc.) and xylazine (10 mg/kg, Vedco Inc.). Each pancreaswas inflated with 3.5 ml cold collagenase (1 mg/ml, type XI, Sigma),excised and immersed for 40 minutes at 37° C. in water bath. Pancreatawere gently vortexed and filtered through 500-micron metal sieve. Thepellet was washed twice in cold HBSS containing 0.5% BSA (Sigma) andreconstituted in RPMI-1640 (Cellgro, Mediatech) supplemented with 10%FCS (Cellgro), 50 IU/ml Penicillin (Cellgro) and 50 μg/ml streptomycin(Cellgro). Islets were collected on a 100-micron nylon cell strainer (BDFalcon), released into a petri dish by rinsing with HBSS (Cellgro,Mediatech) and 0.5% BSA (Sigma) and hand picked under astereomicroscope. For transplantation, 450 islets were thoroughly washedfrom residual FCS in HBSS and 0.5% BSA and mounted on 0.2 ml tip forimmediate transplantation. For in vitro assays islets were left toincubate for 24 hours at 37° C. Islet transplantation was performed intothe left renal subcapsular space. Recipient mice were anesthetized, asdescribed above. An abdominal wall incision was made over the leftkidney. Islets were released into the subcapsular space through apuncture and the opening was sealed by means known in the art. Bloodglucose follow-up was performed 3 times a week from end-tail blood dropusing glucosticks (Roche). (Nanji, S. A. & Shapiro, A. M. Islettransplantation in patients with diabetes mellitus: choice ofimmunosuppression. BioDrugs 18, 315-28 (2004).)

Development of Anti-Human-AAT Antibodies in Mice.

In another exemplary method, in order to evoke specific antibodyproduction against human AAT, mice were injected i.p. with 10 mg humanAAT per 20-gram mouse for four times in intervals of 1 week. Mice wereused in experiments 2 months after last administration. Antibodyproduction was evaluated before transplantation experiments were carriedout.

In one example, assaying for anti-human-AAT antibody levels wasperformed as described in the art. Briefly, mouse sera were kept at −70°C. until assayed for anti-human-AAT levels. Plates were coated withhuman AAT or albumin (2 μg/ml) in PBS at 4° C. overnight, then washedand blocked for 1 hour at 25° C. as described. Negative control serumwas used in addition to test serum. Bound anti-AAT antibody usingstandard TMB substrate solution was measured (Sigma).

Cells.

NIH-3T3 cell line (e.g. ATCC) were cultured. On day of peritonealinoculation, 1×10⁷ cells were freshly collected by trypsinization andwashed with cold PBS. Pellet was resuspended in 1 ml cold PBS forimmediate injection.

Infiltration Experiments.

Peritoneal infiltration was elicited by i.p. injection of 1 mlautoclaved thioglycolate (3% w/v, Sigma) or allogeneic cells (NIH-3T3),together with 0.1 ml saline, human albumin, human AAT or oxidized AAT.Peritoneal lavage was performed at 24 and 48 hours (thioglycolate) or ondays 1-5 (allogeneic cells). For lavage, mice were anesthetized byisoflurane inhalation and injected immediately with 5.5 ml cold PBScontaining 5% FCS and 5 U/ml heparin into the peritoneal cavity. Aftermassaging the abdomen, peritoneal fluid was recovered. Red blood cellswere lysed (RBC lysing buffer, BD PharMingen) and cell counts wereperformed with a hemocytometer. Cells were then isolated. Cells (about1×10⁶/polypropylene vial) were incubated with FcγRIII/II receptor blockantibodies (Table I) for 10 min. Cells were then divided into two groupsand incubated with mAbs for leukocytes and either CD3/NK cells orneutrophil/monocytes/macrophages (Table I) for 30 min. Cells were washedand fixed. The number of cells expressing a particular marker wascalculated by multiplying percentages obtained from flow-cytometry bythe concentration of cells in lavage fluid.

TABLE I Rat Anti-Mouse mAbs Used for Flow Cytometry Purpose mAb (1)Specificity (2) Source Blocking 2.4G2 FcγRIII/II BD PharMingenLeukocytes 30-F11 (APC) CD45 (leukocytes) BD PharMingen MacrophagesF4/80 (PE) F4/80 eBiosciences and (macrophages/ Neutrophils monocytes)RB6-8C5 GR1 (neutrophils/ PharMingen (FITC) monocytes) CD3 DX5 (PE)Pan-NK cells Miltenyi Biotec NK cells 17A2 (FITC) CD3 BD PharMingen TNFαMP6-XT22 (PE) Mouse TNFα eBiosciences MHC class II M5/114.15.2I-A^(b/d), I-E^(d) BD PharMingen (PE) Isotype control Rat IgG1 (PE)eBiosciences

An insulin assay and immunohistochemistry were performed by means knownin the art.

AAT Oxidation by Myeloperoxidase (MPO) System.

In one example, AAT (4 mg/ml) was incubated at 37° C. for 45 minuteswith MPO (1 U/ml, Sigma), H₂O₂ (80 μM, Sigma) and NaCl (2.5 mM) in PBS,pH 7.4, by means known in the art. Reaction was terminated by boilingfor 1 hour followed by filter-centrifugation of the system products. Inthis example, boiling was needed for the inactivation of MPO but thisdid not inactivate AAT (data not shown). Loss of activity of oxidizedAAT was confirmed by elastase activity assay.

Elastase Activity Assay.

In another exemplary method, inhibition of a the serine proteaseelastase was evaluated 30 minutes after co-incubation of AAT or oxidizedAAT with porcine elastase (Sigma) in triplicate, by known methods. Theability of elastase to liberate 4-nitroaniline (A₄₁₀) from SucAla₃-PNAwas determined by kinetic measurement of light absorbance at 410 nm.Activity in the absence of inhibitors was set as 100% at the linearrange of the assay.

Cytokine Assays.

An electrochemiluminescence (ECL) assay as known in the art was used forthe measurement of mouse TNFα and MIP-1α. Briefly, cytokine-specificgoat anti-mouse affinity purified antibodies were labeled with ruthenium(e.g. BioVeris) according to manufacturer's instructions. Biotinylatedpolyclonal anti-mouse antibodies (e.g. R&D Systems) were used. Theamount of TNFα and MIP-1α chemiluminescence was determined using anOrigen Analyzer (BioVeris).

Membrane TNFα.

Membrane TNFα on islet cells was detected by modification of a methodfor the evaluation of membrane TNFα on human PBMC. Briefly, single-cellsuspension of islets was incubated with anti-mTNFα-PE mAb (Table I).Cells were washed with FACS buffer and resuspended in 0.5 ml 2% EM-gradeformaldehyde.

Nitric Oxide Assay.

Nitrite levels in supernatants were determined using Griess reagent(Promega), as previously described (Chan, E. D. & Riches, D. W. Am JPhysiol Cell Physiol 280, C441-50 (2001).

Apoptosis Assay.

The protective effect of AAT on islets may address one of the majorobstacles in islet transplantation today, namely the inadequacy of isletmass and post-isolation islet viability. Freshly isolated human isletsactivate stress signaling pathways and exhibit high rate of apoptosisdue to the process of isolation, necessitating the use of more than oneislet donor per diabetic patient (Nanji, (2004); Abdelli, S. et al.Intracellular stress signaling pathways activated during human isletpreparation and following acute cytokine exposure. Diabetes 53, 2815-23(2004)).

In this example, apoptosis that follows islet isolation is diminishedwhen islets are cultured with AAT (data not shown) and demonstrate thatislets that are cultured with AAT for 24 hours prior to transplantationare able to normalize serum glucose levels of diabetic mice whentransplanted autologously at an otherwise sub-functional mass (data notshown).

AAT Dosage.

Normal human plasma contains 0.8-2.4 mg/ml AAT, with a half life of 5-6days¹. In gene transfer studies in C57BL/6 mice, plasma levels of0.8-1.0 mg/ml were achieved and provided protection from type I diabetesin NOD mice (Song, S. et al Gene Therapy 11, 181-6 (2004)). AATadministered intraperitoneally at 0.3-1.0 mg per mouse protected fromTNFα-induced lethal response, and 0.8 mg AAT protected fromD-galactosamine/LPS induced hepatic injury. Libert, C., et al., JImmunol 157, 5126-9 (1996).

Since AAT levels rise 3- to 4-fold during the acute phase responsel, 2mg per mouse results in plasma levels that do not exceed physiologicallevels.

Statistical Analysis.

Comparisons between groups were analyzed by two-sided t-test or ANOVAfor experiments with more than two subgroups. Results are presented asmean±SEM.

Prolongation of Islet Graft Survival by AAT.

In the present study, administration of clinical grade AAT to micetransplanted with allogeneic islets prolonged graft survival. Inaddition, AAT reduced migration of neutrophils and the subsequentinfiltration of lymphocytes and NK cells in models of peritonitis. AATalso decreased secretion of TNFα and MIP-1α from islets and inhibitedsurface MHC class II expression on CD45+ islet cells in vitro. AAT wasprotective in a model of streptozotocin (STZ)-induced β-cell toxicity.Thus, it appears that AAT monotherapy targets several aspects of anactivated inflammatory immune system, culminating in prolongation ofislet allograft survival.

Effect of AAT on Cell Infiltration.

AAT diminished neutrophil migration into the peritoneum of mice injectedwith either thioglycolate or MHC-incompatible fibroblast cells. Otherstudies demonstrate that AAT inhibits neutrophil infiltration intokidneys during ischemia/reperfusion injury and into lungs followingintratracheal administration of silica. In the present study AATdecreased islet production of MIP-1□ and TNF□, resulting in isletsdeficient in chemotactic capabilities and therefore less immunogenic.The detrimental effect of neutrophils recruited to islets has beenclearly demonstrated.

The involvement of macrophages in islet destruction is critical; theirpresence precedes insulitis in NOD mice and in prediabetic BB rat, andtheir depletion is protective during islet transplantation in rats.Islets are potent recruiters of macrophages; of the 51 gene productsidentified in freshly isolated human islets by cDNA array, expression ofMCP-1 was found to be high. In mice, blockade of MCP-1 prolongs isletallograft survival when combined with a short subtherapeutic course ofrapamycin. Islet allograft rejection is associated with a steadyincrease in intragraft expression of MCP-2, MCP-5, CCLS, CXCL-10 andCXCL9, and the chemokine receptors CCR2, CCR5, CCR1 and CXCR337.Accordingly, CCR2-/-mice and CXCR3-/-mice exhibit prolongation of isletallograft survival. In transplant settings, cytokines that are producedlocally, as TNFα and IL-1β, cause damage to proximal cells independentof antigen recognition, and complement activation is critical for graftsurvival independent of allospecific immunity. The relevance ofmacrophages during early events in islet graft rejection is strengthenedby the identification of CD45, F4/80 and Gr1 positive cells that expressMHC class II in freshly isolated islets. In the presence of AAT, MHCclass II levels were decreased below those of IL-1β/IFNγ-stimulated andunstimulated islets, supporting the idea that the process of isletisolation is sufficient to provoke activation of inflammatory pathwaysin islet cells. In light of the involvement of neutrophils andmacrophages in graft rejection, interference with their functions by AATprovides an unusually non-inflammatory environment for the survival andrecovery of engrafted islets.

As shown in the present study and elsewhere intraperitoneal injection ofallogeneic NIH-3T3 cells evokes infiltration of macrophage andneutrophil on days 1-2 and of CD3+ and NK cells on days 4-5. Theintensity of the latter infiltration was decreased by administration ofAAT prior to allogeneic cell-line injection, but not by administrationof AAT on day 3 (data not shown). In transplant settings, earlynon-specific factors contribute to subsequent specific immune response.It is therefore possible that the decrease in CD3+ and NK cellinfiltration in the present study is secondary to the functional failureof the early innate response. However, regardless of AAT treatment,histological examination of islet grafts demonstrated that theinfiltrate evoked by allogeneic islets consists of neutrophils andlymphocytes. Nevertheless, day 7 infiltrate was diminished inAAT-treated recipients, and, according to day 15 insulinimmunohistochemistry, the infiltrate caused less islet destruction.

AAT Inhibits Release of TNFα.

Supernatants of IL-1β/IFNγ-stimulated islets contained strikingly lessTNFα when incubated with AAT (induction of 100.0%±22.0 mean±SEM at 0mg/ml AAT; 10.2%±11.2 at 0.5 mg/ml and 0.8%±0.1 at 1.0 mg/ml). Instimulated human PBMC, AAT was shown to diminish TNFα release withoutaffecting TNF□-mRNA levels. In mice, accordingly, serum TNFα levels aredecreased in LPS-injected AAT-treated mice. Importantly, treatment ofmice with AAT blocks TNFα-mediated LPS-induced, but not TNF □-inducedlethality in mice. In the present study, cultured mouse splenocytesisolated from thioglycolate-injected mice secreted less TNFα, 48 hoursafter injection of AAT.

In the presence of AAT, membrane TNFα accumulated inIL-1β/IFNγ-stimulated CD45+ islet cells. TNFα is released from the cellsurface of macrophages by the action of TNFα converting enzyme (TACE), ametalloproteinase that cleaves membrane TNFα into the soluble form ofTNFα. Inhibitors of TACE reduce TNFα release and increase the levels ofmembrane TNFα, as demonstrated by FACS analysis. Although the regulationof TACE activity is unclear, there is evidence to suggest thatextracellular proteases are involved: TACE does not require itscytoplasmic domain for its activation, its activity does not depend onthe amount of TACE on the cell surface, co-expression of TACE andtransmembrane TNFα is not sufficient for processing of TNFα and theenzyme is expressed constitutively in various cells. Serpins, such asserpin PN-I52, are suggested to possess extracellular regulatory effectson various surface proteins.

TACE is likely to be relevant for graft rejection since TACE inhibitordecreased injury parameters in a rat model of post-transplant lunginjury. In addition to a decrease in TNFα levels, the study shows lowerexpression of MCP-1 and ICAM-1, and a reduction in neutrophilinfiltration. Similar findings were obtained with both AAT and a broadspectrum metalloproteinase inhibitor in a model of silica inducedneutrophil influx into lungs. However, TACE inhibitor only partiallyreproduced the protective effect of AAT on islet graft survival(preliminary data). Similarly, AAT protection from STZ-inducedhyperglycemia was only partially reproduced by TACE inhibition and byrecombinant p75-TNF-receptor. Despite the fact that locally secretedTNFα is detrimental to islet graft function, there is, to our knowledge,no report that describes protection of islet grafts by neutralization ofTNFα activity. This distinction between AAT and TACE inhibition supportsthe possibility that AAT affects multiple aspects of the immune system,including not only TNFα release but also events that are downstream toTNFα activities.

In one embodiment, it is contemplated that a composition of the presentinvention may include AAT, an analog thereof, a serine protease, TACEinhibitor (TACEi) or any combination thereof. These compositions may beadministered to a subject having or in need of a transplant and or inneed of immunotolerance therapy.

Transplanted islets are stimulated by the process of isolation.

The process of islet isolation initiates in the islets an inflammatorycascade of cytokines and chemokines. Thus, isolated islets contain anintrinsic proinflammatory potential that may affect local host immuneresponses. The mechanism of cytokine-induced islet toxicity is believedto involve expression of inducible nitric oxide synthase and subsequentproduction of nitric oxide (NO) by non-β-cells. In the present study,AAT decreased NO production in IL-1β/IFNγ-treated islets. Accordingly,islet viability was increased in a low NO environment, as attained byeither incubation with a low concentration of stimulators (data notshown) or by introduction of AAT. Insulin induction, which is typicallyincomplete in the presence of cytokines, was intact in the presence ofAAT and cytokines. In vivo, AAT protected islets in mice injected withSTZ, as concluded by lower serum glucose levels. The portion of viableβ-cells was visually assessed by insulin immunohistochemistry and wasproportional to the decrease in serum glucose levels. The protection ofAAT was limited to the initial days that follow STZ administration,suggesting that AAT interferes with NO production and immune activationand not with intracellular DNA alkylation. Freshly isolatednon-stimulated CD45+ islet cells expressed MHC class II, which isinvolved in immune responses against islets. The levels of MHC class IIwere elevated in the presence of IL-1β/IFNγ and decreased in thepresence of AAT. Interestingly, MHCII expression was unaffected by thepresence of TACE (TNF alpha converting enzyme) inhibitor (data notshown), confirming that AAT activities extend beyond those of TACEinhibition.

According to the present study, the activities of AAT are directedagainst multiple components of the innate immune system, culminating ina protective effect on islet graft destruction. Islets in particularexhibited a high degree of protection from inflammatory processes in thepresence of AAT. Pretreatment with AAT prior to islet transplantationmay reduce both islet loss and the immunological response against thegraft.

The terms and expressions that have been employed are used as terms ofdescription and not of limitation, and there is no intention that in theuse of such terms and expressions of excluding any equivalents of thefeatures shown and described or portions thereof, but it is recognizedthat various modifications are possible within the scope of theinvention claimed. Thus, it should be understood that although thepresent invention has been specifically disclosed herein, optionalfeatures, modification and variation of the concepts herein disclosedmay be resorted to by those skilled in the art, and that suchmodifications and variations are considered to be within the scope ofthis invention as defined by the appended claims. In addition, wherefeatures or aspects of the invention are described in terms of Markushgroups, those skilled in the art will recognize that the invention isalso thereby described in terms of any individual member or subgroup ofmembers of the Markush group and that other members of the describedgroups are included but may not be listed.

All publications, patents and patent applications mentioned in thisspecification are indicative of the level of skill of those skilled inthe art to which this invention pertains, and are herein incorporated byreference to the same extent as if each individual publication, patentor patent application was specifically and individually indicated to beincorporated by reference.

Example 9

In one exemplary method to determine whether administration of hAATaffects the severity of GvHD and subsequent mortality from thecondition, three models for GvHD were examined. In the first model, hAATwas administered in a dose-dependent manner in a MHC disparate B6(H-2^(b)) →B6D2F1 (H-2^(b/d)) mouse model and the severity of GvHD wasexamined. Lethally irradiated B6D2F1 mice received BM and splenic Tcells from syngeneic (B6D2F1) or allogeneic (B6) donors. BMT recipientswere injected i.p. with 1, 2, or 4 mg per mouse of either hAAT or humanalbumin as control, as of 2 days prior to transplantation and then everyother day through day 13 after transplantation. This schedule was chosenin order to modulate the inflammatory cascade of acute GvHD, which ismost severe before day 7 after transplantation. In addition, the dose of2 mg per mouse was reportedly effective in allograft rejection andautoimmune models.

As illustrated in FIG. 9A, hAAT administration at 4 mg per mousesignificantly reduced mortality from GvHD when compared with allogeneiccontrols (100% survival vs. 50% survival at day 70, P<0.02). hAATadministration at 1 mg per mouse had only modest effects on mortality,while at 2 mg per mouse hAAT appeared to have prolonged animal survivalAll surviving mice displayed complete donor chimerism (97%+/−2%), asdetermined by fluorescence-activated cell sorter analysis, excludingboth the possibility that graft failure had occurred and that theprotection was the result of mixed chimerism.

To demonstrate that reduction of GvHD by hAAT is strain independent,hAAT was also administered to experimental groups comprised of C3H.SW(H-2^(b))→B6 (H-2^(b)), a MHC matched mouse model of acute GvHD that isdriven by donor CD8⁺ T cells and is directed against minorhistocompatibility antigens. Here, recipient B6 mice were conditionedand then transplanted with either syngeneic (B6) or allogeneic (C3H.SW)donor BM. As shown in FIG. 9B, administration of hAAT at 2 mg per mousesignificantly improved survival when compared with control allogeneichuman albumin-treated mice (78% survival vs. 40% survival at day 60,P<0.03).

In another example, the effect of hAAT in MHC matched minor disparateCD4⁺ dependent but CD8⁺ driven T cell mediated GvHD was examined usingB6 (H-2^(b)) →C3H.SW (H-2^(b)) strain combination. As shown in FIG. 9C,administration of hAAT completely prevented mortality in allogeneicanimals from GvHD when compared to control human albumin-treatedallogeneic animals (100% survival vs. 50% survival at day 40, P<0.02).

Example 10

AAT alters mature donor T effector to Treg ratio after allogeneic BMT.

The expansion of mature donor cytopathic effector T cells (Teffs) andTregs was determined and the ratio of Teffs to Tregs (Teffs:Tregs) wascalculated in allogeneic transplants treated with hAAT. Mice (B6) wereirradiated and transplanted with BM and CD8⁺ T cells from the allogeneicC3H.SW donors. Since expansion of alloreactive T cells peaks at 3-4weeks in this model, splenocytes from recipient animals were isolated atfour weeks and analyzed for donor Teff and Treg cell expansion by donorspecific congeneic markers. (Data not shown). Administration of hAATresulted in reduced expansion of splenic CD8⁺ donor Teffs. In contrast,the expansion of donor Tregs was increased, resulting in the reductionof the Teff:Treg ratio when compared with the control animals. Toexclude the possibility of confounding effects of donor BM derived Tregsin these analyses, and to analyze the kinetics of mature donorTeffs:Tregs, we used B6 GFP⁺ foxp3 knock-in mice as donors for Tregs(GFP⁺ and Ly5.1⁺) and mature Teffs (GFP⁺ and Ly5.1⁺). In a similarmanner, B6 Ly5.2 (CD45.1) used as the source of donor BM are responsiblefor CD45.2⁺ mature Teff (GFP⁻) or Tregs (GFP⁺). These cells were infusedinto the MHC matched but minor disparate C3H. SW animals and theexpansion of the mature Teffs and Tregs was determined.

Since both CD4⁺ and CD8⁺ T cells are responsible for mortality in thismodel, the ratio of both mature donor CD4⁺ Teff and mature CD8⁺ Teffs tothe mature donor GFP⁺ Tregs were analyzed on days 21 and day 28 afterBMT. As shown in FIGS. 10A-10D, the ratio Teffs:Tregs was significantlyaltered for both CD4⁺ and CD8⁺ Teffs on both days 21 and 28. The effectof hAAT was compared to rapamycin, an immunosuppressive agent that hasbeen shown to enhance Tregs in these models. As shown in FIGS. 10C-10D,alteration in Teffs:Tregs ratio by hAAT was similar to the effectsinduced by rapamycin on day 28.

Given the alteration in the expansion and the ratio of the donor Teffsand Tregs cells, we next determined whether hAAT had direct effects ondonor Teff responses in vitro. The proliferation of Treg depleted donorT cells (BALB/c) to allogeneic (B6) BM-derived dendritic cells (DC) wasequivalent regardless of whether the donor T cells were pretreated withhAAT or vehicle (data not shown).

In order to determine the direct impact of hAAT on T cells without aconfounding effect of hAAT on accessory cells, anti-CD3-induced T cellactivation was examined in the presence of hAAT. Consistent withprevious reports addition of AAT did not interfere with T cell responses(data not shown). In addition, hAAT allowed uninterrupted T cell lysisof host-type conA blast cells following priming (57% and 63%, at 50:1E;T ratio, P=NS).

AAT inhibits proinflammatory cytokine release after allogeneic BMT. Thepresence of proinflammatory cytokines has been shown to enhance theexpansion of Teffs while mitigating the responses of Tregs. Inparticular, IL-1β. TNF-α and IL-6 have been shown to play a criticalrole in severity of GvHD. Therefore, alteration of Teffs:Tregs ratio andprotection from GvHD may be a consequence of suppression ofproinflammatory cytokine secretion by hAAT. Mice (B6D2F1) wereirradiated and transplanted with BM and T cells that are eithersyngeneic F1 or allogeneic (B6) donors. Recipient animals were injectedwith either hAAT or albumin at 4 mg per mouse on days −2, 1, and 4 fromtransplantation, and serum samples were analyzed on day +7 fromtransplantation for TNF-α, IL-1β and IL-6. As shown in FIG. 11,administration of hAAT significantly reduced serum levels of all threeproinflammatory cytokines when compared with controlallo-recipients.

Given the lack of direct effect of hAAT monotherapy on donor T cells, wenext reasoned that the reduction in proinflammatory cytokines and GvHDseverity might be the consequence of an effect of hAAT on host antigenpresenting cells (APC). The effect of hAAT on dendritic cells was thusexamined. BM-derived DC from B6 mice were incubated overnight with hAATprior to stimulation with 100 ng/ml LPS for 8 hours. The secretion ofproinflammatory cytokines from AAT-treated DCs was significantly reduced(data not shown). In contrast, the secretion of anti-inflammatorycytokine, IL-10, was significantly enhanced by hAAT when compared withalbumin treated controls. The changes in IL-6 and IL-10 were alsoobserved under similar conditions in host F4/80⁺ macrophages (data notshown).

AAT Inhibits LPS-Induced NF-KB Translocation in DCs

In order to examine a possible mechanism for the reduction ofLPS-induced proinflammatory cytokine secretion by hAAT in BM DCs, NF-KBtranslocation into the nucleus was determined. AAT or human albumin wereadded to BM derived DCs and then stimulated with LPS. NF-κBtranslocation was analyzed by electrophoretic mobility shift assay(EMSA) in the nuclear fraction of cell lysates. Treatment with hAATsignificantly reduced LPSinduced translocation of NF-κB into the nucleuswhen compared with control DCs (data not shown).

In the current study, monotherapy with hAAT reduced proinflammatorycytokines and GvHD mortality in multiple models. The concentrations anddoses of AAT used in the current study are derived and further extendedfrom these reports, according to which 2 mg hAAT per mouse aresufficient to allow islet allograft acceptance (a dose that iscomparable to that used routinely in humans that are deficient in AAT),and 0.5 mg/ml hAAT protects various cell types in vitro from multipleinjuries. Thus, in vivo doses of 1, 2 and 4 mg per mouse were examined,and also tested in vitro concentrations up to 4 mg/ml. The present datademonstrate that exogenous administration of hAAT after allogeneic BMTsuppresses proinflammatory cytokines, alter the ratio of T effectorcells toT regulatory cells and more importantly reduces GvHD severityand related mortality.

MATERIALS and METHODS

Human alpha-1 antitrypsin and albumin: Clinical grade human alpha-1antitrypsin (hAAT, Aralast™) and human albumin were obtained from Baxter(Deerfield, Ill.). Rapamycin was purchased from LC Laboratories (Woburn,Mass.), reconstituted in ethanol at 10 mg/ml and diluted in 5% Tween-80(Sigma) and 5% PEG-400 (Hampton Research, Aliso Viejo, Calif.).Rapamycin was injected at 4 mg/Kg, i.p. daily from 1 day prior to BMTtransplantation.

Mice: Female C57BL/6 (B6, H-2^(b), CD45.2⁺, CD229.1−), C3H.SW (H-2^(b),CD45.2⁺, CD229.1⁺) mice were purchased from the Jackson Laboratories(Bar Harbor, Me.). B6-Ly5.2 (H-2^(b), CD45.1⁺, CD45.2⁻,CD229.1^(−), B)6D2F1 (B6, H-2″, CD45.2⁺, CD229.1⁻) mice were purchasedfrom the Charles River Laboratories (Wilmington, MA). GFP-FoxP3 knock-inmice (GFP-Foxp3, H-2^(b), CD45.1⁺, CD45.2⁺, CD229.1⁻) were provided byDr. Rudensky (University of Washington, Seattle).

Bone marrow transplantation: Bone marrow transplantation was performedas described. Briefly, splenic T cells from donor mice were enriched byMACS cell separation system using anti-CD90.2 microbeads or pan T cellisolation kit (Miltenyi, Auburn, Calif.). Bone marrow T cells weredepleted using anti-CD90.2 microbeads. Recipient mice were irradiated(¹³⁷Cs source) with 10 Gy total body irradiation (TBI) on day −1 andinjected with either syngeneic or allogeneic T cells (1−2×10⁶ or 2×10⁵)along with 4−5×10⁶ T cell-depleted bone marrow cells (TCDBM). Mice werehoused in sterilized microisolator cages and received normal chow andautoclaved hyperchlorinated water for the first 3 weeks after BMT andfiltered water thereafter. Survival was monitored daily, clinical GVHDscore was assessed weekly. Animal studies were approved by theUniversity of Michigan Committee on the Use and Care of Animals.

Bone marrow dendritic cell generation: DCs were generated as described(35). Briefly, Bone marrow cells were isolated from mouse femurs andtibias and cultured in the presence of 20 ng/ml recombinant murineGM-CSF (Peprotech, Rocky Hill, N.J.) for 7 days. CD11c⁺ dendritic cellswere isolated from bone marrow culture by MACS cell separation systemusing CD11c micro beads (Miltenyi).

Flow cytometric analysis: Flow cytometric analysis was performed usingFITC, PE, PerCPCy5.5 or APC-conjugated monoclonal antibodies (mAbs) tomouse CD4 (clone RM4-4), CD229.1 (30C7) (BD Pharmingen, San Jose,Calif.), CD8a (53-6.7), CD25 (PC61.5), CD45.1 (A20), CD45.2 (104),(eBioscience, San Diego, Calif.). Cells were stained, analyzed on aFACSVantage SE (Becton Dickinson, San Jose, Calif.) or C6 cytometer(Accuri Cytometers, Ann Arbor, Mich.) as described.

Electrophoretic mobility shift assay (EMSA): CD11C⁺ DCs were isolatedfrom 16 B6 mice, uniformly divided into 4 dishes (100 mm), treated withhALB (1 mg/ml) or hAAT(1 mg/ml) for 4 hours and then treated with LPS(500 ng/ml) or dileunt for another 3 hours. Nucleic extracts wereincubated with ³²P-ATP-labeled probe specific for NF-κB binding, derivedfrom the class I MHC gene promoter for 30 min at room temperature. Afterreaction, the samples were separated by 5% polyacrylamide gel, dried andvisualized by autoradiography.

Enzyme-linked immunosorbent assay (ELISA): ELISAs for TNFα, IL-1β, IL-6and IL-10 (BD Pharmingen) were performed according to manufacturers'protocol. In vitro suppression assay: CD4⁺CD25⁻ and CD4⁺CD25⁺ T cellswere isolated from spleen cells from BALB/c mice using MACS cellseparation system (Miltenyi). The purity of CD4⁺CD25⁻ and CD4⁺CD25⁺ Tcells was >85%. CD4⁺CD25⁺ T cells were serially diluted from 2×10⁴ to2,500 cells/well and incubated with 2×10⁴ CD4⁺CD25⁻ T cells and 500 or2,500 allogeneic B6 bone marrow-derived dendritic cells for 72, 96 or120 hours. Incorporation of ³H-thymidine (1 μCi/well) by proliferatingcells was measured during the last 12 hours of culture, as described.

Statistical analysis: Survival curves were plotted and compared bylog-rank analysis; P<0.05 was considered statistically significant. Apaired t-test was used to evaluate significant differences betweengroups in in vitro experiments. Data expressed mean±SE.

FIG. 9

(A) B6D2F1 mice were irradiated with 1,000 cGy of total body irradiationin day −1 and transplanted with 5×10⁶ T cell-depleted BM cells and 2×10⁶CD90⁺T cells from either syngeneic F1 or allogeneic B6 donors. Each allorecipient was injected i.p. with either 4 mg hAAT (n=9) or human albumin(n=9) for 6 days from day −2 to day +13. Data shown are combined fromtwo similar experiments. Percent survival after BMT. ▾●vs. ▴, P<0.02 (B)B6 mice were given 1,000 cGy of total body irradiation in day −1 andtransplanted with 5×10⁶ T cell-depleted BM cells and 2×10⁵ CD8⁺T cellsfrom either syngeneic B6 or allogeneic C3H.SW donors. Each allorecipient was injected i.p. with either 2 mg hAAT (n=17) or humanalbumin (n=16) for 6 days from day −2 to day +13 and were monitored forGvHD survival. Data shown are combined from three similar experiments.Percent survival after BMT.▾●vs. ▴, P<0.029.

(C) C3H.SW mice were irradiated as above and transplanted with 4×10⁶ Tcell-depleted BM cells and 1×10⁶ CD90⁺T cells from either syngeneicC3H.SW or allogeneic B6 donors. The allo recipients were injected i.p.with either 2 mg hAAT (n=12) or human albumin (n=15) and were monitoredfor GvHD survival as above. Data shown are combined from two similarexperiments. Percent survival after BMT. ▾●vs. ▴, P<0.019.

FIG. 10 represents tha hAAT alters the ratio of mature donor Teff:Tregcells

C3H.SW mice were irradiated and transplanted with either allogeneicB6GFP⁺Foxp3 knock-in donors. The recipient animals received either hAATor rapamycin or the control vehicle as above. Expansion of mature donor(CD45.2⁺CD45.iCD229.iGFP⁻) CD4 and CD8⁺ effectors and the mature donor(CD45.iCD45.2⁺CD229.iGFP⁺) Tregs were analyzed in the peripheral bloodon day +21 and +28. ratio of CD8⁺ to CD4⁺ FoxP3⁺ T cell absolute numbersof mature donor-derived (CD45.2⁺CD45.iCD229.1⁺). Each point representsone individual mouse (n=2−5/group). (A) CD4⁺: Treg ratio on day +21,vehicle vs. hAAT, P=0.037 (B) CD8⁺: Treg ratio on day +21, vehicle vs.hAAT, P=0.022. (C) CD4⁺: Treg ratio on day +28, vehicle vs. hAAT,P=0.0223 and (D) CD8⁺: Treg ratio on day +28, vehicle vs. hAAT, P=0.0127

FIG. 11 represents injection of hAAT inhibits in vivo proinflammatorycytokine production after BMT B6D2F1 mice were exposed 1,000 cGy oftotal body irradiation and transplanted with 5×10⁶ T cell-depleted BMcells and 2×10⁶ T cells from either allogeneic (B6) or syngeneic(B6D2F1) donors. Each F1 recipient of the allogeneic cells were injectedi.p. with 4 mg hAAT or human albumin on days −2, +1 and +4. Sera fromthe recipient animals (n=4-5 per group) were obtained by on day 7 afterBMT and analyzed for TNFct , IL-1J3 and IL-6 . Albumin treatedallogeneic controls (solid bars) vs. hAAT allogeneic recipients (open,gray bars) for TNFα*, P<0.04. IL-1J3 . Allo **, P<0.03. IL-6 P<0.04.

Example 11

In other embodiments, it was demonstrated that inhibition of IL-32activation by alpha-1 antitrypsin suppresses alloreactivity andincreases survival in an allogeneic murine marrow transplantation model.

Materials and Methods Patients, Sample Collection, and Follow-Up

Patient characteristics, treatment regimens, and clinical outcome datawere collected prospectively and stored in the FHCRC database. Patientswere transplanted for various hematologic malignancies; they were 12-65(median 43) years of age at the time of HCT. Patients receivedcyclosporine or tacrolimus, combined with a short course of methotrexateor mycophenolate mofetil as GVHD prophylaxis. The source of stem cellswere peripheral blood stem cells in 31 patients and bone marrow in 6patients. All patients and controls had given informed consent toparticipate in research studies as required by the Institutional ReviewBoard of the Fred Hutchinson Cancer Research Center (FHCRC).

Patients with acute GVHD. White blood cells (WBC) were collected from 15patients at a median of 24 (range: 18-38) days post-HCT; among these, 10developed acute GVHD and were studied before systemic therapy wasstarted. Five of the 10 acute GVHD patients had serially collectedsamples before onset of GVHD for a total of 15 samples. Eight samples(including 3 sequential ones) were collected from 5 patients who neverdeveloped GVHD during the first 100 days post-HCT. Four of 15 patientswere serologically CMV—, as were 4 of the 15 transplant donors.

Patients with chronic GVHD. PBMC were collected from 22 patients withactive chronic GVHD at a median of 806 (range: 349-5473) days post-HCT;among these, 14 were receiving immunosuppressive therapy, and 8 did not.Among the 22 patients with chronic GVHD, 12 were CMV+, as were 7 of thedonors.

Healthy Controls.

Control samples were collected from 9 healthy individuals, 22-73 (median37) years old.

Cell Separation and Reagents

WBC were separated by Dextran sedimentation (early after HCT when bloodcell counts were low) and PBMC were separated by Ficoll-Hypaque densitygradient centrifugation (in patients with chronic GVHD). RNA wasextracted from WBC and PBMC using Trizol as previously described. cDNAsynthesis was performed from 500 ng of total RNA using InvitrogenSuperscript RT (Invitrogen, Carlsbad, Calif.). Goat polyclonalanti-human IL-32 antibody AF3040, was obtained from R&D Systems(Minneapolis, Minn.), rabbit policlonal antiβ-actin antibody from SantaCruz Biotechnology (Santa Cruz, Calif.) and each used according to themanufacturers' recommended conditions. Concanavalin A was purchased fromSigma-Aldrich Co. (St. Louis, Mo.); Aralast NP (human α-1-antitrypsin),a serum serine-protease inhibitor that blocks the enzymatic activity ofneutrophil elastase, cathespin G, PR3, thrombin, trypsin, andchymotrypsin, was purchased from Baxter (Westlake Village, Calif.).

RNA Interference (RNAi) and Transient Transfection

Stealth siRNA oligonucleotides, specifically designed to silence theexpression of all IL32 isoforms, were obtained from Invitrogen(Carlsbad, Calif.). PBMC from healthy donors (1×10⁶) were electroporatedwith 500 ng of siRNA by nucleofection (Human Cell Nucleofector kit,Program V-024, Amaxa Biosystems, Cologne, Germany).

Human Cytokine Protein Array

After transfection with either scrambled or IL32-specific siRNA, PBMCwere cultured for 96 hours in RPMI 1640 medium, containing 5% fetalbovine serum (FBS), and penicillin/streptomycin (P/S) (50 U/ml and 50μg/ml, respectively), and supernatants were collected. To determine thepresence of various cytokines, we used the human proteome profiler arraymembrane kit Panel A (ARY005; R&D Systems, Minneapolis, Minn.; USA)according to the manufacturer's instructions. Equal volumes (1 ml) ofsupernatant were collected from cultured PBMC and added to the precoatedmembranes of the kit. The dot blot membranes (standardized for loadingcontrol) were analyzed using ImageQuant software (Molecular Dynamics,Sunnyvale, Calif.).

Mixed Leukocyte Cultures (MLCs)

MLCs were used to assess alloreactivity as a simple in vitro model ofGVHD. Human PBMC were suspended in RPMI 1640 medium supplemented with 1%nonessential amino acids; 1% sodium pyruvate; 1% L-glutamine; and 10%heat-inactivated, pooled, normal human serum. One×10⁵ responder cellsand 1×10⁵ irradiated (2,200 cGy) stimulator cells per well wereco-cultured in triplicate in round-bottom 96-well plates for 6 days at37° C. in a humidified 5% carbon dioxide/air atmosphere. MLCs werecarried out either in unmodified medium or with the addition of AAT (atconcentrations of 0.1 to 0.5 mg/ml) or albumin. All final culturevolumes were 200 μl/well. Concanavalin A (Sigma-Aldrich Co. St. Louis,Mo.) was added (4 μg/well) on day 3 to responder cells plated withoutstimulator cells to provide a control for cell proliferation. On day 6,cultures were pulsed with 1 μCi of ³H-thymidine for 18 hours beforeharvesting; ³H-thymidine uptake was measured as the mean counts perminute (cpm) from the three replicates and harvested onto filter paperstrips using a [beta]-scintillation counter (Packard BioScience Company,Meriden, Conn.). Results were expressed as stimulation index (SI)=(meancpm of stimulated cells−mean cpm of nonstimulated cells: mean cpm ofnonstimulated cells).

Markers of Inflammation

Supernatants from MLCs were collected and analyzed for cytokines andinflammatory markers with potential relevance to GVHD, including TNF-α,IL-6 and IL-8, as determined by enzyme-linked immunosorbent assays(ELISA). The probes used included human BAF210 TNF-α, BAF206 IL-6,BAF208 IL-8 (R&D Systems, Minneapolis, Minn.). When inflammatory markerconcentrations were less than the assay detection limit, the sample wasassigned the median value between 0 and the detection limit.

Analysis of Human and Murine Cytokines by Real-Time PCR

RNA was extracted by standard techniques. Applied BiosystemsPre-Designed Gene Expression Assays containing both primers andfluorescent Taq-Man probes were used to determine human or mouse geneexpression.β-actin and GUSB were used as ‘housekeeping’ controls fornormalization of quantitative RNA variation.

Human probes: IL-32, all isoforms (Hs00170403_m1), IL-32β and isoforms(Hs00997068_g1), IL-32α and (Hs00992439_g1), β-actin, (Hs00607939), GUSB(Hs03929099_m1), TNFα (Hs00174128_m1), IL-1β (Hs01555410_m1), PR3(Hs01597752_m1), PAR2 (Hs00173741_m1).

Murine probes: TNFα (Mm00443258_m1), IL-1 (Mm01336189_m1), IL-1Ra(Mm01337566_m1) and PR3 (Mm00478323_m1).

Each 20 μL reaction contained 2.0 γL 10× PCR Buffer without Mg2+, 2.8 μL25 mM MgCl2 (3.5 mM final concentration), 0.4 μL ROX passive referencedye, 0.4 μL 10 mM dNTPs, 1.0 μL ABI primer/probe, and 0.16 μL (0.8 U)Fast Start Taq Polymerase (Roche, Indianapolis, Ind., USA), 8.24 μL H2Oand 5 μL of the cDNA template. All reactions were carried out intriplicate in 384-well plates on an ABI7900HT (Applied Biosystems,Carlsbad, Calif.). For inclusion in the data set, standard deviations ofthe triplicates had to be less than 0.15 CT (cycle threshold).Additionally, we verified that the PCR efficiencies of the ABI assayswere >95% and that the slopes of the linear portion of the amplificationcurves varied by less than 5%

Effect of AAT on GVHD Prevention in an MHC Matched, Minor AntigenDisparate Murine Transplant Model.

C57/BL6J mice (H-2^(b)) (Jackson Laboratory, Bar Harbor, Me.), 10-14weeks old with average body weight of 28 g, received single-dose totalbody irradiation with 1000 cGy followed by intratail vein injection ofT-cell-depleted bone marrow (BM, 5×10⁶ cells), and CD8+ spleniclymphocytes (0.2×10⁶cells) from C3H.SW-H2^(b)/SnJ donors (H-2^(bc))(Jackson Laboratory, Bar Harbor, Me.). BM was T-cell-depleted using theT Cell Isolation Kit II (Milteny Biotec, Auburn, Calif.). CD8+ T-cellswere isolated from splenocytes by positive selection, using MACS CD8+microbeads as directed by the manufacturer (Milteny Biotec, Auburn,Calif.).

Mice in the experimental group were given AAT intra-peritoneally at 3mg/dose, suspended in 125 μl, before irradiation and donor cellinfusion, and every 2 days post-HCT for a total of 10 injections (seeAAT treatment schedule). Mice in the control group were injected, alsointra-peritoneally, with 125 μl of human albumin on the same schedule.Each group consisted of 16 mice. GVHD was assessed by a standard scoringsystem. Body weights were obtained and recorded on day 0 and weeklythereafter. A weekly clinical index was generated by summation of 5criteria scores: percentage of weight change, posture (hunching),activity, fur texture, and skin integrity (maximum score=10). Animalsthat received a score of 6.5 or higher were killed using CO2 euthanasia.Blood samples were collected sequentially for cytokine assays. Todetermine the presence of various cytokines in the two groups of mice,we used the mouse proteome profiler array membrane kit Panel A (ARY006;R&D Systems, Minneapolis, Minn.) according to the manufacturer'sinstructions. Equal volumes (100 μl) of plasma were collected fromindividual animals and added to the precoated membranes of the kit. Thedot blot membranes (standardized for loading control) were analyzedusing ImageQuant software (Molecular Dynamics, Sunnyvale, Calif.). Theexperiments were approved by the Institutional Animal Care and UseCommittee (IACUC) of the FHCRC.

Chimerism Analysis

Chimerism analyses were done on mouse PBMC following separation of bloodon FicollHypaque (density=1.074).¹⁴ Cells at the interface werecollected and washed in phosphate buffered saline by centrifugation. Thecontributions of recipient (C57/BL6J) and donor cells(C3H.SW-H2^(b)/SnJ) to peripheral blood were quantified by fluorescentvariable number of tandem repeat (VNTR) PCR analysis, as described.

Histopathology

At autopsy skin, stomach and small bowel samples were obtained from AATand albumin-treated mice, fixed in 4% paraformaldehyde, and embedded inparaffin before sectioning. The sections were stained withhematoxylin-eosin to assess for inflammatory lesions by lightmicroscopy. The frequencies and severity of inflammatory lesions wereestimated and compared between groups. At least 3 sections from eachorgan were scored.

Statistical Analysis

For gene expression purposes, all values were expressed as the mean±SEM.A Student t test was used to compare continuous variables between twogroups; 1-way analysis of variance (ANOVA) was applied to comparecontinuous variables among three or more groups.

IL-32 expression in MLC. In one example, to determine a potential roleof IL-32 in MLC reactivity, responder cells from MLC were processed forwestern blotting and RNA analysis (data not shown). IL-32 wasupregulated both at the mRNA and protein levels in cells exposed toallogeneic stimulator cells in comparison to autologous controls. Thesupernatants of the same 7-day MLCs revealed high levels of TNF-, IL-6and IL-8 (data not shown).

Repression of IL-32 by siRNA or addition of AAT broadly inhibitsinflammatory mediators. To study the role of endogenous IL-32 in PBMC,IL-32-specific siRNA oligomeres were used, which target each of theIL-32 isoforms as confirmed by BLAST (basic local alignment search tool)alignment. As shown in FIG. 12A, down-regulation of IL-32 by siRNAresulted in a global reduction of cytokine levels in the supernatants,as illustrated by an array of 36 cytokines. The only cytokine that wasup-regulated (by 56% and 60% in two biological duplicates) was I-309, achemokine secreted by regulatory T cells. To determine the impact of AATon IL-32 protein levels human stroma cell line HS5 were used, whichexpresses and secretes IL-32 and can be grown in serum-free medium. Asshown in FIG. 12B, the addition of AAT (at 0.1-1.0 mg/ml) resulted inreductions of endogenous IL-32β and γisoforms. The western blot isrepresentative of three similar experiments.

IL-32 and AAT effect on secreted cytokines in MLC. In another example,the MLCs to which AAT was added at concentrations ranging from 0.1 to0.5 mg/ml were carried out. CD8+ cells sorted from MLCs showed levels ofIL-32β and γisoforms at least 2-fold lower than in the absence of AAT(FIGS. 13A and 13B). Concurrently, there was significant dose-dependantsuppression of the proliferative capacity as determined by ³H thymidineuptake (FIG. 13C), and a 2-fold reduction in TNFαá levels (FIG. 13D).These data demonstrate that AAT had a profound effect on reducingalloreactivity in parallel with inhibition of IL-32 and TNFα production.

IL32 gene expression in blood cells of patients with clinical GVHD. ThenIL32 was examined as a possible biomarker for GVHD by examiningexpression in WBC and PBMC from patients at various time intervals afterHCT. Expression of IL32 in WBC was two-fold higher in patients withacute GVHD than in patients who did not show clinical evidence of GVHD(p<0.02; data not shown); IL32 expression levels in PBMC of patientswith chronic GVHD, untreated (n=8) or treated with steroids,cyclosporine or both (n=14) did not differ from IL32 expression levelsin PBMC of healthy controls (n=9) (p=0.74 and 0.50, respectively) (datanot shown).

AAT abrogates GVHD and reduces mortality in an MHC matched, minorantigen disparate murine transplant model Lethally irradiated (1000 cGy)C57/BL6J (H-2^(b)) mice were injected iv with 5×10⁶ T cell-depleted BMcells and 0.2×10⁶ CD8+ splenic T cells from C3H.SW-H2^(b)/SnJ mice(H-2^(bc)). Recipient mice were given 3 mg of AAT (in 125 μL) on day -1and again on day +2 and every 72 hours for a total of 10 injections(FIG. 14A). Albumin controls received the same volume of human serumalbumin on the same schedule. As shown in FIG. 14B, by day 65 aftertransplantation survival was 80% in AAT-treated mice versus 40% inalbumin treated controls (n=15; p=0.04, log rank). In both albumincontrols and AAT-treated mice C3H.SW-H2^(b)/SnJ donor cells accountedfor more than 95% of cells in peripheral blood (p=0.25) (FIG. 14C). Itis contemplated herein that certain embodiments concern inhibiting IL-32mRNA and/or expression in a subject in need thereof using compositionsdisclosed herein, for example, AAT.

Albumin controls experienced significantly greater weight loss andshowed higher GVHD scores than AAT treated mice (FIG. 14D and 14E). TwoAAT-treated mice that developed signs of gut-GVHD by day 45, i.e. afterdiscontinuation of AAT, showed complete resolution of GVHD uponre-institution of AAT therapy, given every 72 hours, for 4-5 doses.

Histologic examination of albumin-treated mice showed patchy epithelialdamage in the hair follicles and edema (data not shown). The forestomachand duodenum showed patchy lymphocytic infiltration of epithelium anddamage to the glands as evidenced by exocytosis and apoptosis (data notshown). Mice treated with AAT, in contrast, had normal skin and onlyrare areas of infiltration in stomach and duodenum (data not shown).These results indicate that AAT significantly attenuated clinical andhistologic manifestations of GVHD and reduced GVHDrelated morbidity andmortality.

AAT suppresses pro-inflammatory signals and upregulates IL-1Rα in MHCmatched, minor antigen disparate murine transplant recipients.Steady-state levels of IL-1β, TNF-α, and PR3 mRNA on day 21 wassignificantly lower in AATtreated compared to albumin-treated animals(FIG. 15A). Moreover, in a panel of 40 cytokines as illustrated in FIG.7B-D, there was a global suppression of cytokine levels except for IL1Rain the plasma of AAT-treated mice. Also suppressed were, among others,factors such as CXCL13/BLC/BCA-1, a B cell-attracting chemokine1(BCA-1), and CXCL2/MIP-2, known as macrophage inflammatory protein 2α-(MIP2-), a chemokine chemotactic for polymorphonuclear leukocytes.

It was demonstrated that a correlation of IL-32 expression withresponses in MLC and with the manifestations of acute GVHD exists.Although IL-32 is produced locally, the cytokine was readily detected inthe systemic circulation, and IL32 mRNA concentrations in PBMCdiscriminated between patients with and without acute GVHD. Thisobservation raised the possibility that inhibition of IL-32 activationwould interfere with alloactivation and possibly prevent or attenuatethe development and manifestations of GVHD. The present data show thatAAT strongly suppressed CD8+ cell proliferation in allogeneic MLCs, andinhibition of proliferation was associated with suppression of IL-32, aswell as other pro-inflammatory proteins, such as TNFα, IL-8 and IL-6.

Alternatively, the benefit of AAT in that setting may be related toinhibition of IL-32 activation in other tissues, e.g. epithelial cells.The efficacy of AAT in GVHD prevention or treatment is consistent withobservations of the immunosuppressive or immunomodulatory effect of AATin other models. In tune with those findings, over-expression of AAT bygene delivery using recombinant adeno-associated virus significantlyreduced insulinitis and prevented the development of overt hyperglycemiain NOD mice.

Administration of AAT profoundly affected expression of IL-32. The lackof inhibition of IL-1Ra in the context of GVHD prevention is consistentwith previous reports that IL-1Ra inhibited mouse islet allograftrejection and elevated IL-1Ra levels in long-lasting islet allograftsexplanted from AAT treated animals. Thus, compositions disclosed hereincan be combined with other known compositions in order to potentiallyachieve even better inhibition of GvHD and prolonged graft survival.

FIGS. 12A and 12B.—Effect of IL-32 specific siRNA and AAT on expressionof inflammatory mediators. A.) Change in cytokines expression in PBMCtransfected with IL-32-specific or scrambled siRNA (control). Cytokineexpression was assayed using profiler cytokine array (R&D Systems).Cytokine concentrations from siRNA transfected PBMC supernatant areexpressed as percent change in comparison to control supernatants. Shownare changes (mean±SEM) in 29 cytokines. The horizontal line indicates adecrease of 25% in comparison to controls transfected with scrambledsequence. Levels were determined after 72 hours of culture. The membranecontained probes for C5a, ICAM-1, IL-4, IL-13, IL-32α, MIP-11β, CD40ligand, IFN-γ, IL-5, IL-16, IP-10, RANTES, G-CSF, IL-1α, IL-6, IL-17,I-TAC, SDF-1, GM-CSF, IL-1, IL-8, IL-17E, MCP-1, Serpin-E1, GROα,IL-1ra, IL-10, IL-23, MIF, TNFα, I-309, IL-2, IL-12p70, IL-27 MIP-1α,and TREM-1. B) Western blot of protein extract of the human stroma cellline HS5 exposed to vehicle only (veh) or various concentrations of AAT(in serum-free medium). Shown are levels of IL-32β and γ isoforms atconcentrations of ATT between 0.1 and 1 mg/ml.

FIGS. 13A-13D Inhibition of proliferation and TNF secretion in MLC byAAT. (A) Western blot of IL-32β and levels in CD8+ cells from 7-day MLCsunder control conditions and in the presence of AAT (0.3 mg/ml). IL-32and isoforms in the presence of AAT. The western blot is representativeof 3 similar experiments. (B) Expression changes in IL-32 protein levelsin allogeneic MLCs and autologous controls as determined by densitometry(OD) of the same biological experiment. Open columns reflect results inthe absence of AAT; solid columns in the presence of AAT. (C)Proliferation in MLC (as measured by ³H thymidine uptake; CPM,mean±SEM). (D) TNF-ELISA. Secretion of TNF in the presence and absenceof AAT.*indicates p<0.05 (Student t test).

FIGS. 14A-14E. Effect of AAT on GVHD severity and mortality. (A) AATtreatment scheme (see also text). (B) Survival. Survival of AAT-treatedmice versus albumin-treated controls (n=15 each group, p=0.04). (C)Severity of GVHD. GVHD was scored based on percentage of weight loss,skin integrity, posture, mobility, and fur texture. Clinical signs weregraded on a scale of 0 to 2, where 0 was absent, 1 was moderate, and 2was severe, and the individual scores were added up. Shown are GVHDclinical scores for 30 days after transplantation (mean±SEM per timepoint) (D) Change in body weight of transplanted mice over timepost-transplant (mean±SEM; n=15). (E) Donor chimerism. Proportion ofdonor cells among PBMC in AAT-treated (n=6) versus albumin-treated (n=5)mice at day 45 (p=0.25).

FIGS. 15A-15D. Effect of AAT on cytokine RNA and protein expression inPBMC and plasma after transplantation. (A) IL-1Ra, IL-1β, TNF-α, and PR3RNA levels, determined by RT-PCR, in PBMC. Levels in AAT-treated mice(n=6) are expressed relative to levels in albumin treated controls;mean±SEM (n=6) (log2). (B, C and D) Mean±SEM cytokine plasma levels at3, 7 and 10 days after transplantation. Shown is a panel selected from amouse array of 40 cytokines, showing significant changes. Changes incytokine concentration are expressed as percent change compared toalbumin control. The horizontal dotted line indicates anincrease/decrease of 25%.

All of the COMPOSITIONS and METHODS disclosed and claimed herein may bemade and executed without undue experimentation in light of the presentdisclosure. While the COMPOSITIONS and METHODS have been described interms of preferred embodiments, it will be apparent to those of skill inthe art that variation may be applied to the COMPOSITIONS and METHODSand in the steps or in the sequence of steps of the METHODS describedherein without departing from the concept, spirit and scope of theinvention. More specifically, it will be apparent that certain agentswhich are both chemically and physiologically related may be substitutedfor the agents described herein while the same or similar results wouldbe achieved. All such similar substitutes and modifications apparent tothose skilled in the art are deemed to be within the spirit, scope andconcept of the invention as defined by the appended claims.

What is claimed:
 1. A method for treating or preventing acute graftversus host disease in a subject in need thereof, said method comprisingadministering to the subject a composition comprising alpha1-antitrypsin (AAT) or mutant thereof or a carboxyterminal derivativethereof.
 2. The method of claim 1, wherein the composition isadministered to the subject before transplantation, duringtransplantation, after transplantation or combination thereof
 3. Themethod of claim 1, wherein the composition further comprises one or moreanti-transplant rejection agent, anti-inflammatory agent,immunosuppressive agent, immunomodulatory agent, anti-microbial agent,or a combination thereof.
 4. The method of claim 1, whereinadministration occurs within 30 to 40 days after bone marrowtransplantation.
 5. The method of claim 1, wherein administration occursimmediately after bone marrow transplantation.
 6. The method of claim 1,further comprising treating the subject soon after transplantation andpreventing mortality of the subject compared to a subject not treatedwith the composition.
 7. A method for reducing or preventing bone marrowtransplant rejection in a subject having or in need of a bone marrowtransplant, said method comprising: administering bone marrow cells tothe subject; administering one or more dose of a composition comprisingAAT, mutant thereof or carboxyterminal derivative thereof to the subjectwithin two weeks of transplantation of the bone marrow cells; andpreventing GvHD in the subject.
 8. The method of claim 7, wherein thecomposition is AAT.
 9. The method of claim 7, wherein the compositionfurther comprises anti-inflammatory agents.
 10. The method of claim 7,wherein the composition further comprises one or more anti-inflammatoryagent, immunosuppressive agent, immunomodulatory agent, anti-microbialagent, or a combination thereof.
 11. The method of claim 7, wherein thecomposition reduces the production of one or more cytokines.
 12. Themethod of claim 7, wherein the composition further comprises antibodiesto proinflammatory cytokines.
 13. A method for preserving ex vivo bonemarrow cells for transplantation, said method comprising exposing thebone marrow cells to a composition comprising AAT, a mutant thereof orcarboxyterminal derivative thereof.
 14. The method of claim 13, whereinpreserving the bone marrow cells comprises preserving the bone marrowcells prior to implanting the bone marrow cells into a recipient. 15.The method of claim 13, wherein preserving the bone marrow cells isselected from the group consisting of reducing apoptosis, reducingcytokine production, reducing nitric oxide production and a combinationthereof.
 16. A method for reducing need of immunosuppressive agents in asubject having a bone marrow transplantation comprising; identifying asubject having a bone marrow cell transplant and administering atherapeutically effective amount of AAT, AAT mutant or carboxyterminalderivative of AAT to the subject and reducing the need forimmunosuppressive therapy in the subject.
 17. The method of claim 16,wherein the subject is administered the composition before andimmediately after bone marrow transplantation.
 18. The method of claim16, wherein the subject is suffering from a condition selected fromleukemia, severe aplastic anemia, lymphoma, multiple myeloma, immunedeficiency disorder, solid-tumor cancer, breast cancer, ovarian cancerand other conditions.